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[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide.", "This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria LAB , specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012 . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people.", "Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008 . Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella .", "Intracellular bacteria from the genera Mycobacterium . , Listeria . , Shigella . and Salmonella . are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 .", "are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990 . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999 . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 .", "Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals . The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens . . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 .", ". These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004 . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs .", "Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells APCs . After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O LLO; Hly and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997 . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011 . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals.", "Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife and RNA viruses such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus YFV -based vaccines to be used in horses against West Nile virus Draper & Heeney, 2010 . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011 . Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 .", "Amongst them, YFV YF-17D strain was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 Appaiahgari & Vrati, 2010; Rollier et al., 2011 . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus such as modified vaccinia virus Ankara MVA and New York attenuated vaccinia virus NYVAC strains are the most promising vectors Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 . They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability . . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . .", ". The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses . . The adenovirus Ad vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009 . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria.", "They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus HIV -1, influenza, measles, severe acute respiratory syndrome SARS , malaria and tuberculosis M. Saxena and others Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002 . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable of course the minor discomfort that accompanies many vaccinations is normal . Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries Killeen & DiRita, 2000 . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose .", "Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and where appropriate humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose . . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered.", "This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced while the response to the vectored antigen will be a primary response . This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen.", "Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector particularly neutralizing antibody will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . .", "Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 . . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain Bao & Clements, 1991 . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma . reported similar findings.", "Whittle & Verma . reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture.", "These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997 , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b . A slight majority of the studies listed in Table 1 10 versus eight indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . .", "A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain . . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. . indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.", "indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 IL4 response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon IFN -c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig.", "As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen . . Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b .", ". Similar results were reported by Roberts et al. . and Vindurampulle & Attridge 2003a, b . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming Vindurampulle & Attridge, 2003b . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. . immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC.", "Vaccinated mice developed humoral responses to LPS and tetC for the plasmid-bearing vaccines . Animals from all groups including a previously unvaccinated group were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations.", "The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses.", "Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector . . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity . . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used.", ". The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella an intracellular pathogen is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses particularly adaptive responses should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . .", "In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain . . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic as the time between vaccination and infection is short , this may be an option for short-term or perhaps therapeutic vaccination . . Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans.", ". Chickens Gallus gallus are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . .", "Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens . . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong.", "Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges . , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains . . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h . . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated.", ". The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. . indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. .", ". indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. . also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. . in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. .", ". in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. . in which L. monocytogens was used to deliver feline immunodeficiency virus FIV gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals . . Similar findings have been reported by Tvinnereim et al. . and Leong et al. . . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat.", ". However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. . indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens . . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. . also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen.", ". also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 HSV-1 , where the rate of seroprevalence to these viruses is very high 40-45 % and 70 % or more of the US population, respectively Hocknell et al., 2002; Pichla-Gollon et al., 2009 . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 .", "Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens Pichla-Gollon et al., 2009; Pine et al., 2011 . In a large-scale clinical trial STEP of an Ad serotype 5 AdHu5 -based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 . . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen Hocknell et al., 2002; Lauterbach et al., 2005 . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine .", "However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity Brockman & Knipe, 2002 . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine . . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization .", "There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes Kahl et al., 2010; Lasaro & Ertl, 2009 , heterologous prime-boost approaches . , homologous reimmunization . and removing key neutralizing epitopes on the surface of viral capsid proteins Gabitzsch & Jones, 2011; Roberts et al., 2006 . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells DCs . . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 .", ". In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003 . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.", "While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness.", "Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses . . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time.", ". Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent . . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b .", ". A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses Kohler et al., 2000b . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses.", "In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered Vindurampulle & Attridge, 2003b . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains . . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response.", ". For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes Lasaro & Ertl, 2009 . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.", "As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response.", "This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case Saxena et al., 2009 depicted in Fig. 2 . It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered.", "It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection.", "H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies. Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%.", "Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza HPAI strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals.", "Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 .", "Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols. While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 .", "In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II ATII cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus A/Chicken/ Vietnam/0008/04 infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species ROS inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 . The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study.", "The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a \"hit\" for further study. These included: . Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? . Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? . Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? .", "Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? . Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen CEA -related cell adhesion molecule 1 CEACAM1 was chosen as a key gene of interest. CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry .", "CEACAM1 also known as BGP or CD66 is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells DCs . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 . Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short .", "CEACAM1 isoforms Uniprot P13688-1 to -11 can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail i.e. L, long or S, short . The full-length human CEACAM1 protein CEACAM1-4L consists of four extracellular domains one extracellular immunoglobulin variable-region-like IgV-like domain and three immunoglobulin constant region 2-like IgC2-like domains , a transmembrane domain, and a long L cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs ITIMs that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 .", "The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 a related CEACAM family member 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 SH2 -domain containing protein tyrosine phosphatase 1 SHP1 and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 .", "CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody MRG1 to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection.", "In addition, Ceacam1-knockout mice are available for further in vivo infection studies. Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA siRNA -mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA .", "In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection. Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: i uninfected cells treated with 1% DMSO vehicle control ND , ii H5N1-infected cells treated with 1% DMSO HD and iii H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO HA . These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis.", "ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection MOI of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM fragments per kilobase of exon per million fragments mapped > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated q value < 0.05, ≥2-fold change following H5N1 infection ND vs. HD Fig. 1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 .", "1A ; Table S1 . HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism Table 1; Table S2 . In addition, Kyoto Encyclopedia of Genes and Genomes KEGG pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling hsa04668 , Toll-like receptor signaling hsa04620 , cytokine-cytokine receptor interaction hsa04060 and RIG-I-like receptor signaling hsa04622 In the H5N1-infected and apocynin-treated HA group, a large number of genes were also significantly upregulated 509 genes or downregulated 782 genes Fig. 1B ; Table S1 relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig.", "Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated 247 genes, Fig. 1D or downregulated 146 genes, Fig. 1E , a majority of genes did not in fact overlap between the HD and HA groups Fig. 1D , E . This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" .", "This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology GO enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway GO:0060337 , the defense response to virus GO:0009615 , negative regulation of viral processes GO:48525 and the response to stress GO:0006950 Table S2 , \"ND vs. HA Up\" . Genes downregulated by apocynin include those that are involved in cell adhesion GO:0007155 , regulation of cell migration GO:0030334 , regulation of cell proliferation GO:0042127 , signal transduction GO:0007165 and oxidation-reduction processes GO:0055114 Table S2 , \"ND vs. HA Down\" . A total of 623 genes were upregulated following H5N1 infection \"ND vs. HD Up\", Fig. 1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig.", "1F . By overlapping the two lists of genes from \"ND vs. HD Up\" and \"HD vs. HA Down\", 245 genes were shown to be downregulated in the presence of apocynin Fig. 1F . By overlapping three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Up\", 55 genes out of the 245 genes 190 plus 55 genes were present in all three lists Fig. 1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 .", "1G , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2 , Jak-STAT signaling hsa04630; IFNB1, IL15RA, IL22RA1, STAT2 , RIG-I-like receptor signaling hsa04622; DDX58, IFIH1, IFNB1 and Antigen processing and presentation hsa04612; TAP2, TAP1, HLA-DOB Tables S3 and S4 . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the \"ND vs. HA Up\" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells Fig. 1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 .", "1G . The 190 genes include those involved in TNF signaling hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC , cytokine-cytokine receptor interaction hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A , NF-kappa B signaling pathway hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40 and PI3K-Akt signaling hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC Tables S3 and S4 . This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from \"ND vs. HD Up\", \"HD vs. HA Down\" and \"ND vs. HA Down\", 11 genes were found in all three comparisons Fig. 1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig.", "1H . This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells Fig. 1H . Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL a member of the TAM receptor family of receptor tyrosine kinases and TMEM173/STING Stimulator of IFN Genes Table S4 . Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 .", "Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling SOCS , SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production IL6, IFNB1, CXCL10 and CCL5 in A549 cells , which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig.", "IL6 Fig. 2A , IFNB1 Fig. 2B , CXCL10 Fig. 2C , and CCL5 Fig. 2D gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin except for IFNB1 . Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis Fig. 2E . Apocynin treatment further increased SOCS1 mRNA expression Fig. 2E . Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E .", "Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups 4.8-fold vs 4.0-fold Fig. 2E . HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment Fig. 2F . qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" .", "qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection \"ND vs. HD Up\" . The full list of KEGG pathways is presented in Table S3 . of apocynin Fig. 2F . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 .", "Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural NS gene expression was significantly increased Fig. 2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig.", "2G . The reduced cytokine and chemokine production in H5N1-infected ATII cells Fig. 2A-F is unlikely to be associated with lower virus replication. GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 .", "Many of the H5N1-upregulated genes were broadly involved in defense response GO:0006952 , response to external biotic stimulus GO:0043207 , immune system processes GO:0002376 , cytokine-mediated signaling pathway GO:0019221 and type I interferon signaling pathway GO:0060337 Table 1; Table S2 . In addition, many of the H5N1-upregulated genes mapped to metabolic pathways hsa01100 , cytokine-cytokine receptor interaction hsa04060 , Influenza A hsa05164 , TNF signaling hsa04668 or Jak-STAT signaling hsa04630 Table S3 . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above Fig. 1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group.", "1F ; Table S3 . . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method right Y axis normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change left Y axis compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells.", "*p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. G Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A .", "The CEACAM1 transcript was significantly upregulated following H5N1 infection Fig. 3A . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection Fig. 3B . It is also worth noting that more reads were obtained for CEACAM5 >1000 FPKM Fig. 3B than CEACAM1 ~7 FPKM Fig. 3A in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig.", "Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 Fig. 3C and ATII cells Fig. 3D . CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 PR8 virus for 24 or 48 hours when compared to uninfected cells Fig. 3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C .", "3C . No significant difference in CEACAM1 protein levels were observed at various MOIs 2, 5 or 10 or between the 24 and 48 hpi timepoints Fig. 3C . After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection Fig. 3D . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs.", "ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix M gene analyzed by qRT-PCR data not shown . Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi Fig. 3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig.", "3D . HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2 , suggesting a possible role for CEACAM1 in influenza virus pathogenicity Fig. 3D . In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively.", "ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered Fig. 4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B .", "4A . CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells Fig. 4B . Increasing doses of siCEACAM1 10, 15 and 20 pmol did not further downregulate CEACAM1 protein expression in A549 cells Fig. 4B . As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.", "It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L. The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII Fig. 5A and A549 cells Fig. 5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types.", "5B transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells Fig. 5B , but was not affected in siCEACAM1-transfected ATII cells Fig. 5A . Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response.", "Hypercytokinemia or \"cytokine storm\" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII Figs. 5C and 4.8-fold in A549 cells Fig. 5D transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells Fig. 5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses.", "5C,D . Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin.", "These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 .", "Apocynin treatment had both anti-viral Tables S2-S4 5 and pro-viral impact Fig. 2G , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors e.g. DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response.", "DDX58 RIG-I , TLRs, IFIH1 MDA5 was not downregulated Table S1 . Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 .", "H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells Table S4 . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors e.g. IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G .", "IFNB1, MX1, ISG15; Table S1 by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment Fig. 2G . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 . Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 .", "Recognition of intracellular viral RNA by pattern recognition receptors PRRs triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells.", "This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice. NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin HA glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function.", "Given these findings, one could suggest the possibility that upregulation of CEACAM1 to inhibit NK activity may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases e.g. inhibitors for Src, JAK, SHP2 have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 .", "Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity. The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 IRF3 -dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo.", "In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin mTOR to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent. In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection.", "In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes. Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia.", "Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation CSIRO . Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research . . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia.", ". The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution Baxter for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II ATII cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C.", "Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA Gibco twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution HBSS containing elastase 12.9 units/mL; Roche Diagnostics and DNase I 0.5 mg/mL; Roche Diagnostics for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells including macrophages and fibroblasts were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium Gibco and small airway growth medium SAGM medium Lonza containing 5% fetal calf serum FCS and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient 1.089 and 1.040 g/mL . Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C .", "Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS Gibco and 100 units/mL penicillin and 100 µg/mL streptomycin Gibco . ATII cells were plated on polyester Transwell inserts 0.4 μm pore; Corning coated with type IV human placenta collagen 0.05 mg/mL; Sigma at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator 5% CO 2 , 37 °C . Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 .", "A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney MDCK cells were provided by the tissue culture facility of Australian Animal Health Laboratory AAHL , CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium GIBCO and RPMI-1640 medium Invitrogen , respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin GIBCO and maintained at 37 °C, 5% CO 2 . Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 H5N1 was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs.", "Viral stocks of A/Puerto Rico/8/1934 H1N1 PR8 were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories BSL3 at AAHL, CSIRO. Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C.", "Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin BSA containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text.", "Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors different from those used in HiSeq analysis or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone TPCK -treated trypsin Worthington was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis.", "Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 . RNA extraction, quality control QC and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit Qiagen . Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions.", "Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol 10 μL/mL; Gibco . Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set Qiagen , and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer Thermo Scientific ; 2 Sample concentrations were at a minimum of 100 ng/μl; 3 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference.", "As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR qRT-PCR using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome GRCh37, downloaded from Illumina iGenomes as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters.", "Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01.", "Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons \"ND vs. HD Up\", \"ND vs. HD Down\", \"ND vs. HA Up\", \"ND vs. HA Down\", \"HD vs. HA Up\", \"HD vs. HA Down\" . Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .", "Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 . Quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR . mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen .", "Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | . 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix Invitrogen . Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays Applied Biosystems with commercial TaqMan primers and probes, with the exception of the influenza Matrix M gene forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′ 62 . Specific primers 63 Table S5 were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix Bio-Rad according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls.", "The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products 15 μL data not shown . Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad .", "Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit Molecular Probes TM , Invitrogen . Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels Invitrogen , resolved by SDS/PAGE and transferred to PVDF membranes Bio-Rad . Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 ab108397, Abcam followed by goat anti-rabbit HRP-conjugated secondary antibody Invitrogen . Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence ECL Plus Western Blotting Substrate Thermo Scientific followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL GE Healthcare . To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively.", "To use β-actin as a loading control, the same membrane was stripped in stripping buffer 1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2 and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody Cell Signaling . In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software version 1.49J10 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls. Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No.", "Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA siRNA targeting the human CEACAM1 gene siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® or siRNA control siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® using Lipofetamine 3000 ThermoFisher Scientific according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis.", "In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus MOI = 0.01 at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression qRT-PCR between three experimental groups was evaluated using one-way analysis of variance ANOVA followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 .", "The data are shown as means ± standard error of the mean SEM from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows v5.02 . All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO GSE119767; nlm.nih.gov/geo/ ." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality .", "Inflammation of the stomach and the intestines gastroenteritis can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries . In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States .", "In the developed world, viruses are already the most common pathogens causing diarrhea . Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis norovirus was identified in an outbreak of diarrhea in Norwalk California, United States . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis , astrovirus in infantile diarrhea cases , enteric adenoviruses in the feces of children with acute diarrhea , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo.", "Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases diarrhea cases where an enteropathogen could be found , reviewed in the literature . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe .", "Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%-12.7% of all gastroenteritis cases around the globe . Sapovirus outbreaks occur throughout the year and can be foodborne . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection .", "Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature .", "The virus can replicate in human embryonic kidney cells and was detected by electron microscopy EM . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature . Of the 57 identified adenovirus types , only adenoviruses type 40 and 41 are associated with diarrhea . Next to these two types, adenovirus type 52 can also cause gastroenteritis , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear.", "Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis . Aichi virus infection has been shown to elicit an immune response . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea .", "Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus 0.5% and 1.8% was too low to find a significant association with diarrhea . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis . Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive.", "Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature .", "This high sequence diversity has also been observed within particular outbreaks of gastroenteritis , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1.", "Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR ; 2. Random priming-based PCR ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism VIDISCA ; and 4. Sequence-Independent Single Primer Amplification SISPA . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced.", "Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques.", "Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus , Saffold virus , klassevirus/salivirus , polyomavirus , bufavirus , tusavirus , and recovirus . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available.", "Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation prevalence and the ability to cause morbid conditions or disease pathogenesis remains to be determined, as described below also see Table 1 . Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found HMO = human-mink-ovine-like astrovirus ; -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster 55, 57, . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types .", "For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found . In addition an antibody response was observed against some but not all novel astrovirus types . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea .", "Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma HeLa cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples , subsequent case-control studies showed that the virus was not significantly associated with diarrhea . Additionally, in 2008 cosavirus was identified in a patient with diarrhea . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders . In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells .", "In two studies the detection of this virus was associated with diarrhea , while in another study no association with disease was found . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies.", "One year later, STL polyomavirus was found in the stool of a healthy child from Malawi . An antibody response against MX polyomavirus and MW polyomavirus was observed, although MW polyomavirus and STL polyomavirus were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls ; however, because of the low prevalence respectively 0.3% in Thailand and 1.4% in Turkey , no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh .", "Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed.", "Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine , and rhinoviruses , bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus HIV -1 can also be detected in fecal samples . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: .", "Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen . His postulates can be summarized in three points: . The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; . the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and ., after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable.", "If a microbe has fulfilled these three postulates it can be stated that \"the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered\". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described , reviewed in the literature . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing.", "Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses VirCapSeq-VERT which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.", "However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs.", "Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs RT-PCR , in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty.", "In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]