bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice
Authors: Xu Wenting , Zheng Mei , Zhou Feng , Chen Ze
Published: January 2015

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD50]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.
Mapping Antigenic Motifs in the Trypomastigote Small Surface Antigen from Trypanosoma cruzi
Authors: Balouz Virginia , Cámara María de los Milagros , Cánepa Gaspar E. , Carmona Santiago J. , Volcovich Romina , Gonzalez Nicolás , Altcheh Jaime , Agüero Fernán , Buscaglia Carlos A.
Published: January 2015

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule fromTrypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathioneS-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.
Quest for Correlates of Protection against Tuberculosis
Authors: Bhatt Kamlesh , Verma Sheetal , Ellner Jerrold J. , Salgame Padmini
Published: January 2015

A major impediment to tuberculosis (TB) vaccine development is the lack of reliable correlates of immune protection or biomarkers that would predict vaccine efficacy. Gamma interferon (IFN-γ) produced by CD4+T cells and, recently, multifunctional CD4+T cells secreting IFN-γ, tumor necrosis factor (TNF), and interleukin-2 (IL-2) have been used in vaccine studies as a measurable immune parameter, reflecting activity of a vaccine and potentially predicting protection. However, accumulating experimental evidence suggests that host resistance againstMycobacterium tuberculosisinfection is independent of IFN-γ and TNF secretion from CD4+T cells. Furthermore, the booster vaccine MVA85A, despite generating a high level of multifunctional CD4+T cell response in the host, failed to confer enhanced protection in vaccinated subjects. These findings suggest the need for identifying reliable correlates of protection to determine the efficacy of TB vaccine candidates. This article focuses on alternative pathways that mediateM. tuberculosiscontrol and their potential for serving as markers of protection. The review also discusses the significance of investigating the natural human immune response toM. tuberculosisto identify the correlates of protection in vaccination.
Contribution of the BacT/Alert MB Mycobacterium Bottle to Bloodstream Infection Surveillance in Thailand: Added Yield for Burkholderia pseudomallei
Authors: Jorakate Possawat , Higdon Melissa , Kaewpan Anek , Makprasert Sirirat , Yuenprakhon Somkhit , Tawisaid Kittisak , Dejsirilert Surang , Whistler Toni , Baggett Henry C.
Published: January 2015

Community-acquired bloodstream infections cause substantial morbidity and mortality worldwide, but microbiology capacity and surveillance limitations have challenged good descriptions of pathogen distribution in many regions, including Southeast Asia. Active surveillance for bloodstream infections has been conducted in two rural Thailand provinces for >7 years. Blood specimens were divided into two culture bottles, one optimized for aerobic growth (F bottle) and a second for enhanced growth of mycobacteria (MB bottle), and processed with the BactT/Alert 3D system. Because the routine use of MB culture bottles is resource intensive (expensive and requires prolonged incubation), we assessed the added yield of MB bottles by comparing the proportion of pathogens detected by MB versus that by F bottles from 2005 to 2012. Of 63,066 blood cultures, 7,296 (12%) were positive for at least one pathogen; the most common pathogens were
Escherichia coli
(28%),
Burkholderia pseudomallei
(11%),
Klebsiella pneumoniae
(9%), and
Staphylococcus aureus
(6%). Two bottles improved the yield overall, but the added yield attributable to the MB bottles was limited to a few pathogens. In addition to the detection of mycobacteria and some fungi, MB bottles improved the detection of
B. pseudomallei
(27% [MB] versus 8% [F];
P
< 0.0001), with added benefit if therapy was initiated prior to the blood culture. The targeted use of MB bottles is warranted for patients at risk for mycobacterial and fungal infections and for infection with
B. pseudomallei
, a common cause of septicemia in Thailand.
Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis
Authors: Buss Sarah N. , Leber Amy , Chapin Kimberle , Fey Paul D. , Bankowski Matthew J. , Jones Matthew K. , Rogatcheva Margarita , Kanack Kristen J. , Bourzac Kevin M.
Published: January 2015

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic
Escherichia coli
strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens:
Campylobacter
spp.,
Clostridium difficile
(toxin A/B),
Plesiomonas shigelloides
,
Salmonella
spp.,
Vibrio
spp.,
Vibrio cholerae
,
Yersinia enterocolitica
, enteroaggregative
E. coli
, enteropathogenic
E. coli
, enterotoxigenic
E. coli
, Shiga-like toxin-producing
E. coli
(
stx
1
and
stx
2
) (including specific detection of
E. coli
O157),
Shigella
spp./enteroinvasive
E. coli
,
Cryptosporidium
spp.,
Cyclospora cayetanensis
,
Entamoeba histolytica
,
Giardia lamblia
, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (
n
= 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.
Detection of a Transient R292K Mutation in Influenza A/H3N2 Viruses Shed for Several Weeks by an Immunocompromised Patient
Authors: Babady N. Esther , Laplante Jennifer M. , Tang Yi-Wei , St. George Kirsten
Published: January 2015

We describe the case of an immunocompromised patient, positive for influenza A virus (H3N2), in whom the neuraminidase R292K mutation was transiently detected during oseltamivir treatment. The R292K mutation was identified by direct testing in 3 of 11 respiratory specimens collected throughout the patient's illness but in none of the cultures from those specimens.
Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay
Authors: Sevilla Iker A. , Molina Elena , Elguezabal Natalia , Pérez Valentín , Garrido Joseba M. , Juste Ramón A.
Published: January 2015

Mycobacterium tuberculosis
complex,
Mycobacterium avium
, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of
Mycobacterium
genus,
M. avium
subspecies, and
M. tuberculosis
complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (
n
= 38) and nonmycobacterial (
n
= 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (
n
= 57), archival clinical samples (
n
= 233), and strains isolated from various hosts (
n
= 147). The minimum detectable DNA amount per reaction was 50 fg for
M. bovis
BCG and
M. kansasii
and 5 fg for
M. avium
subsp.
hominissuis
. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify
M. avium
and
M. tuberculosis
complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
Defining the Phylogenomics of Shigella Species: a Pathway to Diagnostics
Authors: Sahl Jason W. , Morris Carolyn R. , Emberger Jennifer , Fraser Claire M. , Ochieng John Benjamin , Juma Jane , Fields Barry , Breiman Robert F. , Gilmour Matthew , Nataro James P. , Rasko David A.
Published: January 2015

Shigellae cause significant diarrheal disease and mortality in humans, as there are approximately 163 million episodes of shigellosis and 1.1 million deaths annually. While significant strides have been made in the understanding of the pathogenesis, few studies on the genomic content of the
Shigella
species have been completed. The goal of this study was to characterize the genomic diversity of
Shigella
species through sequencing of 55 isolates representing members of each of the four
Shigella
species:
S. flexneri
,
S. sonnei
,
S. boydii
, and
S. dysenteriae
. Phylogeny inferred from 336 available
Shigella
and
Escherichia coli
genomes defined exclusive clades of
Shigella
; conserved genomic markers that can identify each clade were then identified. PCR assays were developed for each clade-specific marker, which was combined with an amplicon for the conserved
Shigella
invasion antigen, IpaH3, into a multiplex PCR assay. This assay demonstrated high specificity, correctly identifying 218 of 221 presumptive
Shigella
isolates, and sensitivity, by not identifying any of 151 diverse
E. coli
isolates incorrectly as
Shigella
. This new phylogenomics-based PCR assay represents a valuable tool for rapid typing of uncharacterized
Shigella
isolates and provides a framework that can be utilized for the identification of novel genomic markers from genomic data.
Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication
Authors: de Borba Luana , Villordo Sergio M. , Iglesias Nestor G. , Filomatori Claudia V. , Gebhard Leopoldo G. , Gamarnik Andrea V.
Published: January 2015

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new
cis
-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3′ untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.
Constitutive but Not Inducible Attenuation of Transforming Growth Factor β Signaling Increases Natural Killer Cell Responses without Directly Affecting Dendritic Cells Early after Persistent Viral Infection
Authors: Lewis Gavin M. , Macal Monica , Hesser Charles R. , Zuñiga Elina I.
Published: January 2015

Rapid innate responses to viral encounters are crucial to shaping the outcome of infection, from viral clearance to persistence. Transforming growth factor β (TGF-β) is a potent immune suppressor that is upregulated early upon viral infection and maintained during chronic infections in both mice and humans. However, the role of TGF-β signaling in regulating individual cell typesin vivois still unclear. Using infections with two different persistent viruses, murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV; Cl13), in their natural rodent host, we observed that TGF-β signaling on dendritic cells (DCs) did not dampen DC maturation or cytokine production in the early stages of chronic infection with either virusin vivo. In contrast, TGF-β signaling prior to (but not during) chronic viral infection directly restricted the natural killer (NK) cell number and effector function. This restriction likely compromised both the early control of and host survival upon MCMV infection but not the long-term control of LCMV infection. These data highlight the context and timing of TGF-β signaling on different innate cells that contribute to the early host response, which ultimately influences the outcome of chronic viral infectionin vivo.

IMPORTANCEIn vivohost responses to pathogens are complex processes involving the cooperation of many different immune cells migrating to specific tissues over time, but these events cannot be replicatedin vitro. Viruses causing chronic infections are able to subvert this immune response and represent a human health burden. Here we used two well-characterized viruses that are able to persist in their natural mouse host to dissect the role of the suppressive molecule TGF-β in dampening host responses to infectionin vivo. This report presents information that allows an increased understanding of long-studied TGF-β signaling by examining its direct effect on different immune cells that are activated very early afterin vivoviral infection and may aid with the development of new antiviral therapeutic strategies.
Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones
Authors: He Wenqian , Mullarkey Caitlin E. , Duty J. Andrew , Moran Thomas M. , Palese Peter , Miller Matthew S.
Published: January 2015

Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior
in vitro
but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.

IMPORTANCE
Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a “universal” influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of “universal” influenza virus vaccines and therapeutics.
The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Downregulates Fusion F Protein Expression and the F-Specific Antibody Response
In Vivo
Authors: Lingemann Matthias , Surman Sonja , Amaro-Carambot Emérito , Schaap-Nutt Anne , Collins Peter L. , Munir Shirin
Published: January 2015

Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene-end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a readthrough transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to downregulate the expression of F protein by interfering with termination/reinitiation at the M-F gene junction, thus promoting the production of M-F readthrough mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses from which this insert in the M-GE signal was removed were generated. The M-GE mutants exhibited a reduction in M-F readthrough mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wild-type (wt) HPIV3 both
in vitro
and
in vivo
. However, the F-protein-specific serum antibody response in hamsters was increased for the mutants compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation.

IMPORTANCE
The HPIV3 M-GE signal was previously shown to contain an apparent 8-nucleotide insert that was associated with increased synthesis of a readthrough mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that the removal of this insert shifts F gene transcription from readthrough M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in the amount of F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3, the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications for the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by “repairing” the M-GE signal to achieve higher-level F antigen expression, with no effect on attenuation.
On the Stability of Parainfluenza Virus 5 F Proteins
Authors: Poor Taylor A. , Song Albert S. , Welch Brett D. , Kors Christopher A. , Jardetzky Theodore S. , Lamb Robert A.
Published: January 2015

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.
Demographics of Natural Oral Infection of Mosquitos by Venezuelan Equine Encephalitis Virus
Authors: Gutiérrez Serafín , Thébaud Gaël , Smith Darci R. , Kenney Joan L. , Weaver Scott C.
Published: January 2015

The within-host diversity of virus populations can be drastically limited during between-host transmission, with primary infection of hosts representing a major constraint to diversity maintenance. However, there is an extreme paucity of quantitative data on the demographic changes experienced by virus populations during primary infection. Here, the multiplicity of cellular infection (MOI) and population bottlenecks were quantified during primary mosquito infection by Venezuelan equine encephalitis virus, an arbovirus causing neurological disease in humans and equids.
Requirement for Fc Effector Mechanisms in the APOBEC3/Rfv3-Dependent Neutralizing Antibody Response
Authors: Halemano Kalani , Barrett Bradley S. , Heilman Karl J. , Morrison Thomas E. , Santiago Mario L.
Published: January 2015

Antiretroviral neutralizing antibody (NAb) responses are often evaluated in the absence of Fc-dependent immune effectors. In murine Friend retrovirus infection, Apobec3/Rfv3 promotes a potent polyclonal NAb response. Here, we show that the Apobec3/Rfv3-dependent NAb response correlated with virus-specific IgG2 titers and that thein vivoneutralization potency of Apobec3/Rfv3-resistant antisera was dependent on activating Fcγ receptors but not complement. The data strengthen retroviral vaccine strategies aimed at eliciting NAbs that activate specific Fcγ receptors.
Development of Live-Attenuated Arenavirus Vaccines Based on Codon Deoptimization
Authors: Cheng Benson Yee Hin , Ortiz-Riaño Emilio , Nogales Aitor , de la Torre Juan Carlos , Martínez-Sobrido Luis
Published: January 2015

Arenaviruses have a significant impact on public health and pose a credible biodefense threat, but the development of safe and effective arenavirus vaccines has remained elusive, and currently, no Food and Drug Administration (FDA)-licensed arenavirus vaccines are available. Here, we explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse-genetics approaches to rescue a battery of recombinant LCMVs (rLCMVs) encoding CD NPs (rLCMV/NP
CD
) that showed attenuated growth kinetics
in vitro
. Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NP
CD1
and rLCMV/NP
CD2
were highly attenuated
in vivo
but, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NP
CD1
and rLCMV/NP
CD2
were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy, and stability of a CD-based approach for developing live-attenuated vaccine candidates against human-pathogenic arenaviruses.

IMPORTANCE
Several arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections, while antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and is associated with side effects. Here, we describe the generation of recombinant versions of the prototypic arenavirus LCMV encoding codon-deoptimized viral nucleoproteins (rLCMV/NP
CD
). We identified rLCMV/NP
CD1
and rLCMV/NP
CD2
to be highly attenuated
in vivo
but able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated arenavirus vaccines.
Generation of a Transgenic Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease
Authors: Agrawal Anurodh Shankar , Garron Tania , Tao Xinrong , Peng Bi-Hung , Wakamiya Maki , Chan Teh-Sheng , Couch Robert B. , Tseng Chien-Te K.
Published: January 2015

The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg+mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg+mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection.

IMPORTANCESmall and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.
Immune Screening Identifies Novel T Cell Targets Encoded by Antisense Reading Frames of HIV-1
Authors: Berger Christoph T. , Llano Anuska , Carlson Jonathan M. , Brumme Zabrina L. , Brockman Mark A. , Cedeño Samandhy , Harrigan P. Richard , Kaufmann Daniel E. , Heckerman David , Meyerhans Andreas , Brander Christian
Published: January 2015

Cytotoxic-T lymphocyte (CTL) responses to epitopes in alternative HIV reading frames have been reported. However, the extent of CTL responses to putative proteins encoded in antisense reading frames is unknown. Using sequence alignments and computational approaches, we here predict five potential antisense HIV proteins and characterize common CTL responses against them. Results suggest that antisense-derived sequences are commonly transcribed and translated and could encode functional proteins that contain important targets of anti-HIV cellular immunity.
A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B
env
Gene
Authors: Pugach Pavel , Ozorowski Gabriel , Cupo Albert , Ringe Rajesh , Yasmeen Anila , de Val Natalia , Derking Ronald , Kim Helen J. , Korzun Jacob , Golabek Michael , de los Reyes Kevin , Ketas Thomas J. , Julien Jean-Philippe , Burton Dennis R. , Wilson Ian A. , Sanders Rogier W. , Klasse P. J. , Ward Andrew B. , Moore John P.
Published: January 2015

Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and
in vitro
properties of a new SOSIP.664 trimer derived from a subtype B
env
gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.

IMPORTANCE
The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A
env
gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.
Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs
Authors: Pauly Matthew D. , Lauring Adam S.
Published: January 2015

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus.

IMPORTANCE
Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation.
A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication
Authors: Hsu Shih-Feng , Su Wen-Chi , Jeng King-Song , Lai Michael M. C.
Published: January 2015

Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication.

IMPORTANCE
Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection.
Normal saline flushes performed once daily maintain peripheral intravenous catheter patency: a randomised controlled trial
Authors: Schreiber Silvana , Zanchi Chiara , Ronfani Luca , Delise Anna , Corbelli Alessandra , Bortoluzzi Rosamaria , Taddio Andrea , Barbi Egidio
Published: January 2015

Objective

Recent evidence supports the use of normal saline flushes in place of heparin to maintain the patency of peripheral intravenous locks (IVLs); however, there are no data regarding the recommended flush frequency.

Study design

This was an open, non-inferiority, randomised controlled trial. Children with IVLs, aged 1–17 years, were randomly assigned to receive saline flushing every 12 h (group A) or every 24 h (group B). The main outcome was the maintenance of catheter patency.

Results

Four hundred patients were randomised; 198 subjects were analysed in the 12 h group and 199 in the 24 h group (three patients were lost at follow-up). Occlusion occurred in 15 children (7.6%) in group A versus 9 (4.5%) in group B (p=0.21). The difference in catheter patency was +3.1% in favour of the 24 h group (95% CI −1.6% to 7.7%), showing the non-inferiority of the 24 h procedure (the non-inferiority margin was set at −4%). Catheter-related complications were not different between the two groups (12.1% in group A vs 9.5% in group B; p=0.42).

Conclusions

A flushing procedure with one flush per day allows maintenance of catheter patency without an increase in catheter-related complications. We propose a simplification of the flushing procedure with only one flush per day, thereby reducing costs (materials use and nursing time), labour and unnecessary manipulation of the catheters which can cause distress in younger children and their parents.

Trial registration number

The study is registered in the international database ClinicalTrial.gov under registration number NCT02221024.
Patterns of relapse in extrapulmonary small cell carcinoma: retrospective analysis of outcomes from two cancer centres
Authors: Gennatas S , Noble J , Stanway S , Gunapala R , Chowdhury R , Wotherspoon A , Benepal T , Popat S
Published: January 2015

Objectives

We conducted a retrospective review of patients with extrapulmonary small cell carcinomas (EPSCCs) to explore the distribution, treatments, patterns of relapse and outcomes by primary site.

Setting

We have reviewed the outcomes of one of the largest data sets of consecutive patients with EPSCC identified from two major cancer centres.

Participants

Consecutive patients with a histopathological diagnosis of EPSCC from the two institutions were retrospectively identified.

Primary and secondary outcome measures

Outcomes were evaluated including stage at presentation, treatments given, sites of relapse, time to distant relapse, progression-free survival and overall survival (OS).

Results

From a total 159 patients, 114 received first-line chemotherapy, 80.5% being platinum-based. Response rate was 48%. Commonest primary sites were genitourinary and gynaecological. 44% of patients presented with metastatic disease. 55.9% relapsed with liver the commonest site, whereas only 2.5% developed brain metastases. Median OS was 13.4 months for all patients, 7.6 months and 19.5 months for those with metastatic and non-metastatic disease, respectively. Gynaecological and head and neck patients had significantly better OS compared to gastrointestinal patients.

Conclusions

EPSCCs demonstrate high response rates to chemotherapy and high rates of distant metastases. Primary sites may influence prognosis, and survival is optimal with a radical strategy. Brain metastases are rare and we therefore do not recommend prophylactic cranial irradiation.
Can probiotic yogurt prevent diarrhoea in children on antibiotics? A double-blind, randomised, placebo-controlled study
Authors: Fox Michael J , Ahuja Kiran D K , Robertson Iain K , Ball Madeleine J , Eri Rajaraman D
Published: January 2015

Objective

To estimate the efficacy of a probiotic yogurt compared to a pasteurised yogurt for the prevention of antibiotic-associated diarrhoea in children.

Design and setting

This was a multisite, randomised, double-blind, placebo-controlled clinical trial conducted between September 2009 and 2012. The study was conducted through general practices and pharmacies in Launceston, Tasmania, Australia.

Participants and interventions

Children (aged 1–12 years) prescribed antibiotics, were randomised to receive 200 g/day of either yogurt (probiotic) containingLactobacillus rhamnosusGG (LGG),Bifidobacterium lactis(Bb-12) andLactobacillus acidophilus(La-5) or a pasteurised yogurt (placebo) for the same duration as their antibiotic treatment.

Outcomes

Stool frequency and consistency were recorded for the duration of treatment plus 1 week. Primary outcome was stool frequency and consistency, classified at different levels of diarrhoea severity. Due to the small number of cases of diarrhoea, comparisons between groups were made using Fisher's exact analysis.

Results

72 children commenced and 70 children (36 placebo and 34 probiotic) completed the trial. There were no incidents of severe diarrhoea (stool consistency ≥6, ≥3 stools/day for ≥2 consecutive days) in the probiotic group and six in the placebo group (Fisher's exact p=0.025). There was also only one episode of minor diarrhoea (stool consistency ≥5, ≥2 stools/day for ≥2 days in the probiotic group compared to 21 in the placebo group (Fisher's exact p<0.001). The probiotic group reported fewer adverse events (1 had abdominal pain, 1 vomited and 1 had headache) than the placebo group (6 had abdominal pain, 4 had loss of appetite and 1 had nausea).

Conclusions

A yogurt combination of LGG, La-5 and Bb-12 is an effective method for reducing the incidence of antibiotic-associated diarrhoea in children.

Trial registration number

Australian New Zealand Clinical Trials Registry ACTRN12609000281291
The African, Caribbean and European (ACE) Pathways to Care study: a qualitative exploration of similarities and differences between African-origin, Caribbean-origin and European-origin groups in pathways to care for psychosis
Authors: Ferrari Manuela , Flora Nina , Anderson Kelly K , Tuck Andrew , Archie Suzanne , Kidd Sean , McKenzie Kwame
Published: January 2015

Objectives

This paper reports on a qualitative exploration of the reasons for differences in pathways to care and duration of untreated psychosis (DUP) in the African, Caribbean and European (ACE) Pathways to Care study from the perspective of respondents to the study and their families.

Setting

Ontario, Canada.

Participants

Thirty-four participants in total. Twenty-five young people who had experienced a first episode of psychosis and nine family members. Participants were part of the ACE Pathways to Care study.

Design

We implemented six focus groups. Furthermore, we implemented four in-depth interviews with two African-origin young women, one Caribbean-origin woman, and one European-origin woman with lived experience of psychosis.

Results

Factors that influenced help-seeking delays across the three groups were: personal awareness of symptoms, family members’ knowledge of psychotic symptoms and knowledge of mental health services. Youth and their family members described how stigma played a key role in pathways to care by stopping them from asking for help. The way in which stigma operated on the three groups’ members, from feeling ashamed to feeling guilty for their mental illnesses, helped to explain differences in DUP between the groups. Guilt feelings emerged as a prominent theme among members from the African and Caribbean groups and it was not discussed in the European focus group. Delay in entering into first-episode psychosis programmes was also influenced by the stigma perceived by young people in healthcare settings. This had an impact on the therapeutic relationships, disclosure of symptoms and overall trust in the healthcare system.

Conclusions

The findings of this paper suggest that stigma, especially internalised stigma, may operate in different ways in European-origin, African-origin and Caribbean-origin groups. These findings could inform the development of more equitable services for people in early stages of psychosis.