title: Air-Liquid Interface Method To Study Epstein-Barr Virus Pathogenesis in Nasopharyngeal Epithelial Cells

text: Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation. EBV also infects epithelial cells, predominantly resulting in a lytic infection, which may contribute to EBV transmission from saliva. In the nasopharynx, EBV infection can lead to the clonal expansion of a latently infected cell and the development of nasopharyngeal carcinoma (NPC). The mechanisms governing EBV pathogenesis in nasopharyngeal epithelium are largely unknown. An advanced understanding would depend on a physiologically relevant culture model of polarized airway epithelium. The recent application of the organotypic raft culture in keratinocytes has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication. In this study, the adaptation of an air-liquid interface (ALI) culture method using transwell membranes was explored in an EBV-infected NPC cell line. In the EBV-infected NPC HK1 cell line, ALI culture resulted in the completion of EBV reactivation, with global induction of the lytic cascade, replication of EBV genomes, and production of infectious progeny virus. We propose that the ALI culture method can be widely adopted as a physiologically relevant model to study EBV pathogenesis in polarized nasal epithelial cells. IMPORTANCE Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo. In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.


title: Febrile seizures: an overview

text: BACKGROUND: Febrile seizures are the most common neurologic disorder in childhood. Physicians should be familiar with the proper evaluation and management of this common condition. OBJECTIVE: To provide an update on the current understanding, evaluation, and management of febrile seizures. METHODS: A PubMed search was completed in Clinical Queries using the key terms ‘febrile convulsions’ and ‘febrile seizures’. The search strategy included meta-analyses, randomized controlled trials, clinical trials, observational studies, and reviews. RESULTS: Febrile seizures, with a peak incidence between 12 and 18 months of age, likely result from a vulnerability of the developing central nervous system to the effects of fever, in combination with an underlying genetic predisposition and environmental factors. The majority of febrile seizures occur within 24 hours of the onset of the fever. Febrile seizures can be simple or complex. Clinical judgment based on variable presentations must direct the diagnostic studies which are usually not necessary in the majority of cases. A lumbar puncture should be considered in children younger than 12 months of age or with suspected meningitis. Children with complex febrile seizures are at risk of subsequent epilepsy. Approximately 30–40% of children with a febrile seizure will have a recurrence during early childhood. The prognosis is favorable as the condition is usually benign and self-limiting. Intervention to stop the seizure often is unnecessary. CONCLUSION: Continuous preventative antiepileptic therapy for the prevention of recurrent febrile seizures is not recommended. The use of intermittent anticonvulsant therapy is not routinely indicated. Antipyretics have no role in the prevention of febrile seizures.


title: De novo design of isopeptide bond-tethered triple-stranded coiled coils with exceptional resistance to unfolding and proteolysis: implication for developing antiviral therapeutics

text: Isopeptide bond-tethered triple-stranded coiled coils of HIV-1 gp41 N-terminal heptad repeat (NHR) peptides have been designed with de novo auxiliaries to guide site-directed trimerized cross-linking. The presence of isopeptide bridges in the rationally designed trimerization motifs provides extraordinary stability to withstand thermal and chemical denaturation. As a result, these ultra-stable and well-folded trimeric coiled coils direct and yield proteolysis-resistant and remarkably potent N-peptide chimeric trimers with HIV-1 fusion inhibitory activities in the low nanomolar range, much more effective than the corresponding unstructured N-peptide monomers and reaching the potency of clinically used T20 peptide (enfuvirtide). Thus, these isopeptide bond-crosslinked de novo coiled coils may also be used as attractive scaffolds for isolating NHR-trimers in other class I enveloped viruses for therapeutic intervention. Furthermore, this isopeptide bridge-tethering strategy could be extendable to the construction of ultra-stable proteins interfering with certain biological processes.


title: A Note on the Risk of Infections Invading Unaffected Regions

text: We present two probabilistic models to estimate the risk of introducing infectious diseases into previously unaffected countries/regions by infective travellers. We analyse two distinct situations, one dealing with a directly transmitted infection (measles in Italy in 2017) and one dealing with a vector-borne infection (Zika virus in Rio de Janeiro, which may happen in the future). To calculate the risk in the first scenario, we used a simple, nonhomogeneous birth process. The second model proposed in this paper provides a way to calculate the probability that local mosquitoes become infected by the arrival of a single infective traveller during his/her infectiousness period. The result of the risk of measles invasion of Italy was of 93% and the result of the risk of Zika virus invasion of Rio de Janeiro was of 22%.


title: Functional Characterization of Plasmodium falciparum Surface-Related Antigen as a Potential Blood-Stage Vaccine Target

text: Plasmodium falciparum erythrocyte invasion is a multistep process that involves a spectrum of interactions that are not well characterized. We have characterized a 113-kDa immunogenic protein, PF3D7_1431400 (PF14_0293), that possesses coiled-coil structures. The protein is localized on the surfaces of both merozoites and gametocytes, hence the name Plasmodium falciparum surface-related antigen (PfSRA). The processed 32-kDa fragment of PfSRA binds normal human erythrocytes with different sensitivities to enzyme treatments. Temporal imaging from initial attachment to internalization of viable merozoites revealed that a fragment of PfSRA, along with PfMSP1(19,) is internalized after invasion. Moreover, parasite growth inhibition assays showed that PfSRA P1 antibodies potently inhibited erythrocyte invasion of both sialic acid–dependent and –independent parasite strains. Also, immunoepidemiological studies show that malaria-infected populations have naturally acquired antibodies against PfSRA. Overall, the results demonstrate that PfSRA has the structural and functional characteristics of a very promising target for vaccine development.


title: Acute disseminated encephalomyelitis: a call to the clinicians for keeping this rare condition on clinical radar

text: Acute disseminated encephalomyelitis is a rare disease of central nervous system, which can present with a variety of clinical manifestations. That is why first attack of ADEM, in particular remains a diagnostic puzzle. Early anticipation and diagnosis is important for better outcomes. We present a case of acute disseminated encephalomyelitis which initially had atypical clinical features with cough, expectoration, fever and later manifested strange neurological features, diagnosed to be a case of acute disseminated encephalomyelitis based on radio-imaging.


title: Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus

text: To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.


title: Expanding Patient Access to Investigational New Drugs: Overview of Intermediate and Widespread Treatment Investigational New Drugs, and Emergency Authorization in Public Health Emergencies

text: Individual patients with life-threatening or severely debilitating diseases can petition the U.S. Food and Drug Administration (FDA) through their physicians to have expanded access (EA) to drugs that are in clinical trials but have not reached full FDA approval (the “single-patient” investigational new drug [IND] application). Additionally, recent state and federal laws—so-called “right to try legislation”—allow patients to approach drug companies directly for access prior to FDA approval. While these pathways provide potential access for individual patients to investigational drugs, different EA pathways permit entire groups of certain patients to access investigational drugs prior to FDA approval. This review focuses on special categories of EA INDs intended for multiple patients—the intermediate-group IND and the widespread-treatment IND—as well as emergency authorization for use of investigational drugs and biological products (e.g., vaccines) in public health emergencies.


title: Frequency of shedding of respiratory pathogens in horses recently imported to the United States

text: BACKGROUND: Imported horses that have undergone recent long distance transport might represent a serious risk for spreading infectious respiratory pathogens into populations of horses. OBJECTIVE: To investigate the frequency of shedding of respiratory pathogens in recently imported horses. ANIMALS: All imported horses with signed owner consent (n = 167) entering a USDA quarantine for contagious equine metritis from October 2014 to June 2016 were enrolled in the study. METHODS: Prospective observational study. Enrolled horses had a physical examination performed and nasal secretions collected at the time of entry and subsequently if any horse developed signs of respiratory disease during quarantine. Samples were assayed for equine influenza virus (EIV), equine herpesvirus type‐1, −2, −4, and −5 (EHV‐1, −2, −4, −5), equine rhinitis virus A (ERAV), and B (ERBV) and Streptococcus equi subspecies equi (S. equi) using quantitative PCR (qPCR). RESULTS: Equine herpesviruses were detected by qPCR in 52% of the study horses including EHV‐2 (28.7%), EHV‐5 (40.7%), EHV‐1 (1.2%), and EHV‐4 (3.0%). Clinical signs were not correlated with being qPCR‐positive for EHV‐4, EHV‐2, or EHV‐5. None of the samples were qPCR‐positive for EIV, ERAV, ERBV, and S. equi. The qPCR assay failed quality control for RNA viruses in 25% (46/167) of samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs of respiratory disease were poorly correlated with qPCR positive status for EHV‐2, −4, and −5. The importance of γ‐herpesviruses (EHV‐2 and 5) in respiratory disease is poorly understood. Equine herpesvirus type‐1 or 4 (EHV‐1 or EHV‐4) were detected in 4.2% of horses, which could have serious consequences if shedding animals entered a population of susceptible horses. Biosecurity measures are important when introducing recently imported horses into resident US populations of horses.


title: Nucleocapsid protein-based vaccine provides protection in mice against lethal Crimean-Congo hemorrhagic fever virus challenge

text: Crimean-Congo hemorrhagic fever (CCHF) is an acute, often fatal viral disease characterized by rapid onset of febrile symptoms followed by hemorrhagic manifestations. The etiologic agent, CCHF orthonairovirus (CCHFV), can infect several mammals in nature but only seems to cause clinical disease in humans. Over the past two decades there has been an increase in total number of CCHF case reports, including imported CCHF patients, and an expansion of CCHF endemic areas. Despite its increased public health burden there are currently no licensed vaccines or treatments to prevent CCHF. We here report the development and assessment of the protective efficacy of an adenovirus (Ad)-based vaccine expressing the nucleocapsid protein (N) of CCHFV (Ad-N) in a lethal immunocompromised mouse model of CCHF. The results show that Ad-N can protect mice from CCHF mortality and that this platform should be considered for future CCHFV vaccine strategies.


title: Intrathecal B Cells in MS Have Significantly Greater Lymphangiogenic Potential Compared to B Cells Derived From Non-MS Subjects

text: Although B cell depletion is an effective therapy of multiple sclerosis (MS), the pathogenic functions of B cells in MS remain incompletely understood. We asked whether cerebrospinal fluid (CSF) B cells in MS secrete different cytokines than control-subject B cells and whether cytokine secretion affects MS phenotype. We blindly studied CSF B cells after their immortalization by Epstein-Barr Virus (EBV) in prospectively-collected MS patients and control subjects with other inflammatory-(OIND) or non-inflammatory neurological diseases (NIND) and healthy volunteers (HV). The pilot cohort (n = 80) was analyzed using intracellular cytokine staining (n = 101 B cell lines [BCL] derived from 35 out of 80 subjects). We validated differences in cytokine production in newly-generated CSF BCL (n = 207 BCL derived from subsequent 112 prospectively-recruited subjects representing validation cohort), using ELISA enhanced by objective, flow-cytometry-based B cell counting. After unblinding the pilot cohort, the immortalization efficiency was almost 5 times higher in MS patients compared to controls (p < 0.001). MS subjects' BCLs produced significantly more vascular endothelial growth factor (VEGF) compared to control BCLs. Progressive MS patients BCLs produced significantly more tumor necrosis factor (TNF)-α and lymphotoxin (LT)-α than BCL from relapsing-remitting MS (RRMS) patients. In the validation cohort, we observed lower secretion of IL-1β in RRMS patients, compared to all other diagnostic categories. The validation cohort validated enhanced VEGF-C production by BCL from RRMS patients and higher TNF-α and LT-α secretion by BCL from progressive MS. No significant differences among diagnostic categories were observed in secretion of IL-6 or GM-CSF. However, B cell secretion of IL-1β, TNF-α, and GM-CSF correlated significantly with the rate of accumulation of disability measured by MS disease severity scale (MS-DSS). Finally, all three cytokines with increased secretion in different stages of MS (i.e., VEGF-C, TNF-α, and LT-α) enhance lymphangiogenesis, suggesting that intrathecal B cells directly facilitate the formation of tertiary lymphoid follicles, thus compartmentalizing inflammation to the central nervous system.


title: Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement

text: Influenza viruses replicate within the nucleus of the host cell. This uncommon RNA virus trait provides influenza with the advantage of access to the nuclear machinery during replication. However, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. The aim of this review is to present the current mechanistic understanding for how IAVs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the IAV infection process.


title: Wobbling Forth and Drifting Back: The Evolutionary History and Impact of Bacterial tRNA Modifications

text: Along with tRNAs, enzymes that modify anticodon bases are a key aspect of translation across the tree of life. tRNA modifications extend wobble pairing, allowing specific (“target”) tRNAs to recognize multiple codons and cover for other (“nontarget”) tRNAs, often improving translation efficiency and accuracy. However, the detailed evolutionary history and impact of tRNA modifying enzymes has not been analyzed. Using ancestral reconstruction of five tRNA modifications across 1093 bacteria, we show that most modifications were ancestral to eubacteria, but were repeatedly lost in many lineages. Most modification losses coincided with evolutionary shifts in nontarget tRNAs, often driven by increased bias in genomic GC and associated codon use, or by genome reduction. In turn, the loss of tRNA modifications stabilized otherwise highly dynamic tRNA gene repertoires. Our work thus traces the complex history of bacterial tRNA modifications, providing the first clear evidence for their role in the evolution of bacterial translation.


title: Identification and characterisation of the CD40-ligand of Sigmodon hispidus

text: Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses.


title: Overview of three influenza seasons in Georgia, 2014–2017

text: BACKGROUND: Influenza epidemiological and virologic data from Georgia are limited. We aimed to present Influenza Like Illness (ILI) and Severe Acute Respiratory Infection (SARI) surveillance data and characterize influenza viruses circulating in the country over three influenza seasons. METHODS: We analyzed sentinel site ILI and SARI data for the 2014–2017 seasons in Georgia. Patients’ samples were screened by real-time RT-PCR and influenza viruses isolated were characterized antigenically by haemagglutination inhibition assay and genetically by sequencing of HA and NA genes. RESULTS: 32% (397/1248) of ILI and 29% (581/1997) of SARI patients tested were positive for influenza viruses. In 2014–2015 the median week of influenza detection was week 7/2015 with B/Yamagata lineage viruses dominating (79%); in 2015–2016—week 5/2016 was the median with A/H1N1pdm09 viruses prevailing (83%); and in 2016–2017 a bimodal distribution of influenza activity was observed—the first wave was caused by A/H3N2 (55%) with median week 51/2016 and the second by B/Victoria lineage viruses (45%) with median week 9/2017. For ILI, influenza virus detection was highest in children aged 5–14 years while for SARI patients most were aged >15 years and 27 (4.6%) of 581 SARI cases died during the three seasons. Persons aged 30–64 years had the highest risk of fatal outcome, notably those infected with A/H1N1pdm09 (OR 11.41, CI 3.94–33.04, p<0.001). A/H1N1pdm09 viruses analyzed by gene sequencing fell into genetic groups 6B and 6B.1; A/H3N2 viruses belonged to genetic subclades 3C.3b, 3C.3a, 3C.2a and 3C.2a1; B/Yamagata lineage viruses were of clade 3 and B/Victoria lineage viruses fell in clade1A. CONCLUSION: In Georgia influenza virus activity occurred mainly from December through March in all seasons, with varying peak weeks and predominating viruses. Around one third of ILI/ SARI cases were associated with influenza caused by antigenically and genetically distinct influenza viruses over the course of the three seasons.


title: Greater Microbial Translocation and Vulnerability to Metabolic Disease in Healthy Aged Female Monkeys

text: Monkeys demonstrate gastrointestinal barrier dysfunction (leaky gut) as evidenced by higher biomarkers of microbial translocation (MT) and inflammation with ageing despite equivalent health status, and lifelong diet and environmental conditions. We evaluated colonic structural, microbiomic and functional changes in old female vervet monkeys (Chlorocebus aethiops sabeus) and how age-related leaky gut alters responses to Western diet. We additionally assessed serum bovine immunoglobulin therapy to lower MT burden. MT was increased in old monkeys despite comparable histological appearance of the ascending colon. Microbiome profiles from 16S sequencing did not show large differences by age grouping, but there was evidence for higher mucosal bacterial loads using qPCR. Innate immune responses were increased in old monkeys consistent with higher MT burdens. Western diet challenge led to elevations in glycemic and hepatic biochemistry values only in old monkeys, and immunoglobulin therapy was not effective in reducing MT markers or improving metabolic health. We interpret these findings to suggest that ageing may lead to lower control over colonization at the mucosal surface, and reduced clearance of pathogens resulting in MT and inflammation. Leaky gut in ageing, which is not readily rescued by innate immune support with immunoglobulin, primes the liver for negative consequences of high fat, high sugar diets.


title: Managing emerging transnational public health security threats: lessons learned from the 2014 West African Ebola outbreak

text: BACKGROUND: Pandemics pose significant security/stability risks to nations with fragile infrastructures. We evaluated characteristics of the 2014 West African Ebola outbreak to elucidate lessons learned for managing transnational public health security threats. METHODS: We used publically available data to compare demographic and outbreak-specific data for Guinea, Sierra Leone, and Liberia, including key indicator data by the World Health Organization. Pearson correlation statistics were calculated to compare country-level infrastructure characteristics with outbreak size and duration. RESULTS: Hospital bed density was inversely correlated with longer EVD outbreak duration (r = − 0.99). Country-specific funding amount allocations were more likely associated with number of incident cases than the population at-risk or infrastructure needs. Key indicators demonstrating challenges for Guinea included: number of unsafe burials, percent of EVD-positive samples, and days between symptom onset and case hospitalization. Sierra Leone’s primary key indicator was the number of districts with ≥1 security incident. Liberia controlled their outbreak before much of the key-indicator data were collected. CONCLUSION: Many of the country-level factors, particularly the WHO key indicators were associated with controlling the epidemic. The infrastructure of countries affected by communicable diseases should be assessed by international political and public health leaders.


title: Oral edible plant vaccine containing hypoallergen of American cockroach major allergen Per a 2 prevents roach-allergic asthma in a murine model

text: BACKGROUND: American cockroaches (Periplaneta americana) are an important indoor allergen source and a major risk factor for exacerbations and poor control of asthma. We previously reported that allergen components from American cockroaches exhibit varying levels of pathogenicity. Sensitization to major American cockroach allergen, Per a 2, correlated with more severe clinical phenotypes among patients with allergic airway diseases. MATERIALS AND METHODS: In this study, we examined whether oral plant vaccine-encoding full-length Per a 2 clone-996 or its hypoallergenic clone-372 could exert a prophylactic role in Per a 2-sensitized mice. The cDNAs coding Per a 2–996 and Per a 2–372 were inserted into TuMV vector and expressed in Chinese cabbage. Adult female BALB/c mice were fed with the cabbage extracts for 21 days and subsequently underwent two-step sensitization with recombinant Per a 2. RESULTS: Per a 2-specific IgE measured by in-house ELISA in the sera of Per a 2-372-treated groups were significantly lower than in the control groups after allergen challenge but not the Per a 2-996-treated group. Moreover, Per a 2–372 vaccine markedly decreased airway hyper-responsiveness and infiltration of inflammatory cells into the lungs, as well as reduced mRNA expression of IL-4 and IL-13 in comparison with the control mice. CONCLUSION: Our data suggest that oral administration of edible plant vaccine encoding Per a 2 hypo-allergen may be used as a prophylactic strategy against the development of cockroach allergy.


title: Mapping Oil Spills from Dual-Polarized SAR Images Using an Artificial Neural Network: Application to Oil Spill in the Kerch Strait in November 2007

text: Synthetic aperture radar (SAR) has been widely used to detect oil-spill areas through the backscattering intensity difference between oil and background pixels. However, since the signal is similar to that produced by other phenomena, positive identification can be challenging. In this study we developed an algorithm to effectively analyze large-scale oil spill areas in SAR images by focusing on optimizing the input layer to artificial neural network (ANN) through removal the factor of lowering the accuracy. An ANN algorithm was used to generate probability maps of oil spills. Highly accurate pixel-based data processing was conducted through false or un-detection element reduction by normalizing the image or applying a non-local (NL) means filter and median filter to the input neurons for ANN. In addition, the standard deviation of co-polarized phase difference (CPD) was used to reduce false detection from the look-alike with weak damping effect. The algorithm was validated using TerraSAR-X images of an oil spill caused by stranded oil tanker Volganefti-139 in the Kerch Strait in 2007. According to the validation results of the receiver operating characteristic (ROC) curve, the oil spill was detected with an accuracy of about 95.19% and un-detection or false detection by look-alike and speckle noise was greatly reduced.


title: Tick-Borne Flaviviruses and the Type I Interferon Response

text: Flaviviruses are globally distributed pathogens causing millions of human infections every year. Flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. Mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response.


title: CXCL9 promotes prostate cancer progression through inhibition of cytokines from T cells

text: Chemokines have been demonstrated to serve an important role in a variety of diseases, particularly in tumor progression. There have been numerous studies that have reported that T cells serve major roles in tumor progression. However, the function of CXC motif chemokine ligand 9 (CXCL9) in prostate cancer remains unknown. The present study aimed to investigate the role of CXCL9 in prostate cancer. A prostate cancer mouse model was generated by treating C57/BL-6 and B6.Cg-Selplgtm1Fur/J mice with 3,2′-dimethyl 4-aminobiphenyl (DMAB). Hematoxylin and eosin staining detected the histopathological alterations of mouse prostate tissues. Immunohistochemistry (IHC) staining determined cell proliferation of the mice. Flow cytometry was used to detect the alterations of T cells in C57+DMAB or CXCL9+DMAB mice. Immunofluorescence revealed that there was positive expression of interleukin-6 (IL-6) and transforming growth factor (TGF)-β in the mouse tissues. The survival rates of C57+DMAB and CXCL9+DMAB mice was analyzed. The association of CXCL9 expression and clinical stages was also evaluated. Results revealed that prostate cancer pathology and cell proliferation in CXCL9+DMAB mice were significantly greater compared with the C57+DMAB mice. Compared with C57+DMAB mice, the number of T cells in peripheral blood and spleen of CXCL9+DMAB mice was significantly reduced. IHC demonstrated that the expression of IL-6 and TGF-β was significantly downregulated in the CXCL9+DMAB mice. The survival rate of CXCL9+DMAB mice was significantly decreased compared with the C57+DMAB mice. In addition, reverse transcription-quantitative polymerase chain reaction analysis demonstrated that CXCL9 mRNA expression in clinical samples was positively associated with clinical pathological stages of prostate cancer. In conclusion, CXCL9 may promote prostate cancer progression via inhibition of cytokines from T cells.


title: The Convergence of High-Consequence Livestock and Human Pathogen Research and Development: A Paradox of Zoonotic Disease

text: The World Health Organization (WHO) estimates that zoonotic diseases transmitted from animals to humans account for 75 percent of new and emerging infectious diseases. Globally, high-consequence pathogens that impact livestock and have the potential for human transmission create research paradoxes and operational challenges for the high-containment laboratories that conduct work with them. These specialized facilities are required for conducting all phases of research on high-consequence pathogens (basic, applied, and translational) with an emphasis on both the generation of fundamental knowledge and product development. To achieve this research mission, a highly-trained workforce is required and flexible operational methods are needed. In addition, working with certain pathogens requires compliance with regulations such as the Centers for Disease Control (CDC) and the U.S. Department of Agriculture (USDA) Select Agent regulations, which adds to the operational burden. The vast experience from the existing studies at Plum Island Animal Disease Center, other U.S. laboratories, and those in Europe and Australia with biosafety level 4 (BSL-4) facilities designed for large animals, clearly demonstrates the valuable contribution this capability brings to the efforts to detect, prepare, prevent and respond to livestock and potential zoonotic threats. To raise awareness of these challenges, which include biosafety and biosecurity issues, we held a workshop at the 2018 American Society for Microbiology (ASM) Biothreats conference to further discuss the topic with invited experts and audience participants. The workshop covered the subjects of research funding and metrics, economic sustainment of drug and vaccine development pipelines, workforce turnover, and the challenges of maintaining operational readiness of high containment laboratories.


title: Application of Gold Nanoparticle to Plasmonic Biosensors

text: Gold nanoparticles (GNPs) have been widely utilized to develop various biosensors for molecular diagnosis, as they can be easily functionalized and exhibit unique optical properties explained by plasmonic effects. These unique optical properties of GNPs allow the expression of an intense color under light that can be tuned by altering their size, shape, composition, and coupling with other plasmonic nanoparticles. Additionally, they can also enhance other optical signals, such as fluorescence and Raman scattering, making them suitable for biosensor development. In this review, we provide a detailed discussion of the currently developed biosensors based on the aforementioned unique optical features of GNPs. Mainly, we focus on four different plasmonic biosensing methods, including localized surface plasmon resonance (LSPR), surface-enhanced Raman spectroscopy (SERS), fluorescence enhancement, and quenching caused by plasmon and colorimetry changes based on the coupling of GNPs. We believe that the topics discussed here are useful and able to provide a guideline in the development of novel GNP-based biosensors in the future.


title: The Differential Effects of Alcohol and Nicotine-Specific Nitrosamine Ketone on White Matter Ultrastructure

text: AIMS: The chronic consumption of alcohol is known to result in neurodegeneration and impairment of cognitive function. Pathological and neuroimaging studies have confirmed that brain atrophy in alcoholics is mainly due to widespread white matter (WM) loss with neuronal loss restricted to specific regions, such as the prefrontal cortex. Neuroimaging studies of cigarette smokers also suggest that chronic inhalation of tobacco smoke leads to brain atrophy, although the neurotoxic component is unknown. As a high proportion of chronic alcoholics also smoke cigarettes it has been hypothesized that at least some alcohol-related brain damage is due to tobacco smoke exposure. METHODS: 39 Long Evans rats were subjected to 8 weeks exposure to alcohol and/or 5 weeks co-exposure to nicotine-specific nitrosamine ketone (NNK), a proxy for tobacco smoke. Their frontal WM was then assayed with transmission electron microscopy. RESULTS: NNK and ethanol co-exposure had a synergistic effect in decreasing myelinated fibre density. Furthermore, NNK treatment led to a greater reduction in myelin sheath thickness than ethanol whereas only the ethanol-treated animals showed a decrease in unmyelinated fibre density. CONCLUSION: These data suggest that NNK causes WM degeneration, an effect that is exacerbated by alcohol, but unlike alcohol, it has little impact on the neuronal components of the brain.


title: The usefulness of hand washing during field training to prevent acute respiratory illness in a military training facility

text: Hand washing plays a key role in preventing respiratory infection in many clinical settings. However, its effectiveness in preventing acute respiratory illness (ARI) during field training in military training facilities has been not studied. A quasi-interventional study was performed to evaluate the prevalence of ARIs over 4 weeks in a Korean army training center in South Korea from January 2009 to February 2009. A total of 1291 recruits participating in military training for 4 weeks were randomly distributed to 2 battalions (one with 631 and the other with 660). After noticing there is a difference between the 2 battalions in terms of the development of ARIs at the end of 2 weeks of training, we conducted interviews with the battle commanders to determine factors that may be related to one battalion having a higher incidence of ARI. Thereafter, we performed an intervention, which consists of instructing the battalion having a higher incidence of ARI to implement field hand washing from the third week. Following the intervention, we compared the cumulative rate of ARI during 4 weeks of training. The interviews revealed that there were no major differences between the 2 battalions in terms of the training schedules, living environments, or indoor hand washing methods. However, there was difference in terms of hand washing during field training for the first 2 weeks; whereas one battalion (the early hand washing group) implemented hand washing during field training starting in the first week, the other battalion did not implement hand washing for the first 2 weeks but instead began in the third week (the late hand washing group). The cumulative incidence rate of ARI during 4 weeks of training was significantly lower in the early hand washing group (13.0%, 95% confidence interval [CI]: 10.6%–15.9%) than in the late hand washing group (28.0%, 95% CI, 24.7%–31.5%). Our study suggests that outdoor hand washing during field training may be an effective precaution for reducing ARI incidence among recruits participating in military training.


title: Laboratory epidemiology of respiratory viruses in a large children's hospital: A STROBE-compliant article

text: Acute respiratory tract infection (ARTI) is the most common causes of outpatient visit and hospital admission for children. The study aimed to report epidemiological data on respiratory viruses in a university-affiliated children's hospital. The study was a retrospective study conducted in a university affiliated children's hospital from 2016 May to 2017 April. The results of all nasopharyngeal swab and sputum samples sent for the test for respiratory viruses (adenovirus, influenza A, influenza B, and respiratory syncytial virus) were extracted from the electronic healthcare records. Clinical characteristics were compared between groups with positive versus negative results for respiratory viruses. Multivariable regression models were employed by including age, gender, type of sample (swab vs sputum), source (emergency department vs others), and season to explore the independent factors associated with positive results for respiratory viruses. A total of 34,961 samples were identified during the study period. A total of 3102 (8.9%) samples were positive for adenovirus, 2811 (8.0%) were positive for influenza A, 3460 (9.9%) were positive for influenza B, and 4527 (13.0%) were positive for respiratory syncytial virus. The positive rate of adenovirus was highest in April (50.8%), and lowest in November (3%). The absolute number of positive samples for adenovirus was highest in June (n = 587) and April (n = 544). For the test of influenza A, age was independently associated with positive result. With 1 year increase in age, the odds of positive result increased by 12% (odds ratio [OR]: 1.12; 95% confidence interval [CI]: 1.11–1.13; P < .001). As compared with the autumn, the summer showed significantly lower rate of positive for RSV (OR: 0.49; 95% CI: 0.38–0.62; P < .001), whereas the winter had higher risk of positive result (OR: 3.88; 95% CI: 3.37–4.50; P < .001). The study reported epidemiological data on the prevalence of respiratory viruses in a large tertiary care children's hospital. Age, gender, type of sample, source, and season were associated with the positive rates for respiratory viruses.


title: Exogenous lipoid pneumonia: an important cause of interstitial lung disease in infants

text: Exogenous lipoid pneumonia (ELP), an important cause of interstitial lung disease, often goes unrecognized. We conducted a retrospective study of children with histologically confirmed ELP at Red Cross Children’s Hospital, South Africa. Twelve children of Zimbabwean heritage aged 2.1–10.8 months were identified between 2012 and 2017. Repeated oral administration of plant‐based oil for cultural reasons was reported by 10 of 11 caregivers. Cough (12/12), tachypnoea (11/12), hypoxia (9/12), and diffuse alveolar infiltrates on chest radiography (12/12) were common at presentation. Chest computed tomography revealed ground‐glass opacification with lower zone predominance (9/9) and interlobular septal thickening (8/9). Bronchoalveolar lavage specimens appeared cloudy/milky, with abundant lipid‐laden macrophages and extracellular lipid on Oil‐Red‐O staining (12/12), with polymicrobial (6/12) and Mycobacterium abscessus (2/12) co‐infection. Antibiotics, systemic corticosteroids, and therapeutic lavage were interventions in all eight and five patients, respectively. Clinicians should consider ELP in children with non‐resolving pneumonia in settings with similar practices.


title: A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial

text: BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)—compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.


title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

text: Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been studied previously, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens is hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm to host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.


title: In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype

text: Following injury, mesenchymal repair cells are activated to function as leader cells that modulate wound healing. These cells have the potential to differentiate to myofibroblasts, resulting in fibrosis and scarring. The signals underlying these differing pathways are complex and incompletely understood. The ex vivo mock cataract surgery cultures are an attractive model with which to address this question. With this model we study, concurrently, the mechanisms that control mesenchymal leader cell function in injury repair within their native microenvironment and the signals that induce this same cell population to acquire a myofibroblast phenotype when these cells encounter the environment of the adjacent tissue culture platform. Here we show that on injury, the cytoskeletal protein vimentin is released into the extracellular space, binds to the cell surface of the mesenchymal leader cells located at the wound edge in the native matrix environment, and supports wound closure. In profibrotic environments, the extracellular vimentin pool also links specifically to the mesenchymal leader cells and has an essential role in signaling their fate change to a myofibroblast. These findings suggest a novel role for extracellular, cell-surface–associated vimentin in mediating repair-cell function in wound repair and in transitioning these cells to a myofibroblast phenotype.


docid: mvdtoplq

title: Virus-Receptor Interactions: The Key to Cellular Invasion

text: Virus-receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses utilize elegant strategies to attach to one or multiple receptors, overcome the plasma membrane barrier, enter, and access the necessary host cell machinery. The viral attachment protein can be viewed as the “key” that unlocks host cells by interacting with the “lock” – the receptor – on the cell surface, and these lock-and-key interactions are critical for viruses to successfully invade host cells. Many common themes have emerged in virus receptor utilization within and across virus families demonstrating that viruses often target particular classes of molecules in order to mediate these events. Common viral receptors include sialylated glycans, cell adhesion molecules (CAMs) such as immunoglobulin superfamily (IgSF) members and integrins, and phosphatidylserine (PtdSer) receptors. The redundancy in receptor usage suggests that viruses target particular receptors or “common locks” to take advantage of their cellular function and also suggests evolutionary conservation. Due to the importance of initial virus interactions with host cells in viral pathogenesis and the redundancy in viral receptor usage, exploitation o f these strategies would be an attractive target for new antiviral therapeutics.


title: Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

text: Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.


title: Heterosubtypic Protections against Human-Infecting Avian Influenza Viruses Correlate to Biased Cross-T-Cell Responses

text: Against a backdrop of seasonal influenza virus epidemics, emerging avian influenza viruses (AIVs) occasionally jump from birds to humans, posing a public health risk, especially with the recent sharp increase in H7N9 infections. Evaluations of cross-reactive T-cell immunity to seasonal influenza viruses and human-infecting AIVs have been reported previously. However, the roles of influenza A virus-derived epitopes in the cross-reactive T-cell responses and heterosubtypic protections are not well understood; understanding those roles is important for preventing and controlling new emerging AIVs. Here, among the members of a healthy population presumed to have previously been infected by pandemic H1N1 (pH1N1), we found that pH1N1-specific T cells showed cross- but biased reactivity to human-infecting AIVs, i.e., H5N1, H6N1, H7N9, and H9N2, which correlates with distinct protections. Through a T-cell epitope-based phylogenetic analysis, the cellular immunogenic clustering expanded the relevant conclusions to a broader range of virus strains. We defined the potential key conserved epitopes required for cross-protection and revealed the molecular basis for the immunogenic variations. Our study elucidated an overall profile of cross-reactivity to AIVs and provided useful recommendations for broad-spectrum vaccine development.


title: Label‐Free Quantitative Proteomic Analysis of Differentially Expressed Membrane Proteins of Pulmonary Alveolar Macrophages Infected with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Its Attenuated Strain

text: Significant differences exist between the highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) and its attenuated pathogenic (AP) strain in the ability to infect host cells. The mechanisms by which different virulent strains invade host cells remain relatively unknown. In this study, pulmonary alveolar macrophages (PAMs) are infected with HP‐PRRSV (HuN4) and AP‐PRRSV (HuN4‐F112) for 24 h, then harvested and subjected to label‐free quantitative MS. A total of 2849 proteins are identified, including 95 that are differentially expressed. Among them, 26 proteins are located on the membrane. The most differentially expressed proteins are involved in response to stimulus, metabolic process, and immune system process, which mainly have the function of binding and catalytic activity. Cluster of differentiation CD163, vimentin (VIM), and nmII as well as detected proteins are assessed together by string analysis, which elucidated a potentially different infection mechanism. According to the function annotations, PRRSV with different virulence may mainly differ in immunology, inflammation, immune evasion as well as cell apoptosis. This is the first attempt to explore the differential characteristics between HP‐PRRSV and its attenuated PRRSV infected PAMs focusing on membrane proteins which will be of great help to further understand the different infective mechanisms of HP‐PRRSV and AP‐PRRSV.


title: Correlation of microbiological yield with radiographic activity on chest computed tomography in cases of suspected pulmonary tuberculosis

text: BACKGROUND: Little is known about the correlation between microbiological yield and radiographic activity, on chest computed tomography (CT), in suspected pulmonary tuberculosis (PTB) cases, despite CT being widely used, clinically. METHODS: We used multicenter retrospective data, obtained from medical records, focusing on the diagnostic performance for definite PTB. We categorized patients into four groups, by radiographic activity: definitely active, probably active, indeterminate activity, and probably inactive. RESULTS: Of the 650 patients included, 316 had culture-confirmed PTB; 190 (29.2%), 323 (49.7%), 70 (10.8%), and 67 (10.3%) were classified into the definitely active, probably active, indeterminate activity, and probably inactive groups, respectively. The corresponding observed culture rates for CT radiographic activity were 61.6%, 60.7%, 4.3% and 0%, respectively. When not only culture rates but TB-PCR and histological results were taken into consideration as definite PTB, it showed 66.6%, 67.2%, 14.3%, and 0% of each CT radiographic activity, respectively. Regarding the diagnostic performance for definite PTB, radiographic activity displayed high sensitivity (97.1%, 95% confidence interval (CI), 94.6–98.5) and negative predictive values (92.7%, 95% CI, 86.6–96.2), considered definitely and probably active PTB. Apart from PTB, other etiologies, according to radiographic activity, were predominantly respiratory infections such as bacterial pneumonia and non-tuberculous mycobacterial infection. CONCLUSIONS: Radiographic activity showed good diagnostic performance, and can be used easily in clinical practice. However, clinicians should consider other possibilities, because radiologic images do not confirm microbiological PTB.


title: Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies

text: As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.


title: Autophagy enhances the replication of Peste des petits ruminants virus and inhibits caspase-dependent apoptosis in vitro

text: Peste des petits ruminants (PPR) is an acute and highly contagious disease in small ruminants that causes significant economic losses in developing countries. An increasing number of studies have demonstrated that both autophagy and apoptosis are important cellular mechanisms for maintaining homeostasis, and they participate in the host response to pathogens. However, the crosstalk between apoptosis and autophagy in host cells during PPRV infection has not been clarified. In this study, autophagy was induced upon virus infection in caprine endometrial epithelial cells (EECs), as determined by the appearance of double- and single-membrane autophagy-like vesicles, LC3-I/LC3-II conversion, and p62 degradation. We also found that PPRV infection triggered a complete autophagic response, most likely mediated by the non-structural protein C and nucleoprotein N. Moreover, our results suggest that autophagy not only promotes the replication of PPRV in EECs but also provides a potential mechanism for inhibiting PPRV-induced apoptosis. Inhibiting autophagosome formation by wortmannin and knocking down the essential autophagic proteins Beclin-1 and ATG7 induces caspase-dependent apoptosis in EECs in PPRV infection. However, inhibiting autophagosome and lysosome fusion by NH(4)Cl and chloroquine did not increase the number of apoptotic cells. Collectively, these data are the first to indicate that PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication and maturity in host cells.


title: A Recombinant Newcastle Disease Virus (NDV) Expressing S Protein of Infectious Bronchitis Virus (IBV) Protects Chickens against IBV and NDV

text: Infectious bronchitis virus (IBV) causes a highly contagious respiratory, reproductive and urogenital tract disease in chickens worldwide, resulting in substantial economic losses for the poultry industry. Currently, live-attenuated IBV vaccines are used to control the disease. However, safety, attenuation and immunization outcomes of current vaccines are not guaranteed. Several studies indicate that attenuated IBV vaccine strains contribute to the emergence of variant viruses in the field due to mutations and recombination. Therefore, there is a need to develop a stable and safe IBV vaccine that will not create variant viruses. In this study, we generated recombinant Newcastle disease viruses (rNDVs) expressing the S1, S2 and S proteins of IBV using reverse genetics technology. Our results showed that the rNDV expressing the S protein of IBV provided better protection than the rNDV expressing S1 or S2 protein of IBV, indicating that the S protein is the best protective antigen of IBV. Immunization of 4-week-old SPF chickens with the rNDV expressing S protein elicited IBV-specific neutralizing antibodies and provided complete protection against virulent IBV and virulent NDV challenges. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV.


title: Development of a short course on management of critically ill patients with acute respiratory infection and impact on clinician knowledge in resource‐limited intensive care units

text: BACKGROUND: The 2009 influenza A (H1N1) pandemic caused surges of patients in intensive care units (ICUs) in resource‐limited settings. Several Ministries of Health requested clinical management guidance from the World Health Organization (WHO), which had not previously developed guidance regarding critically ill patients. OBJECTIVE: To assess the acceptability and impact on knowledge of a short course about the management of critically ill patients with acute respiratory infections complicated by sepsis or acute respiratory distress syndrome delivered to clinicians in resource‐limited ICUs. METHODS: Over 4 years (2009‐2013), WHO led the development, piloting, implementation and preliminary evaluation of a 3‐day course that emphasized patient management based on evidence‐based guidelines and used interactive adult‐learner teaching methodology. International content experts (n = 35) and instructional designers contributed to development. We assessed participants’ satisfaction and content knowledge before and after the course. RESULTS: The course was piloted among clinicians in Trinidad and Tobago (n = 29), Indonesia (n = 38) and Vietnam (n = 86); feedback from these courses contributed to the final version. In 2013, inaugural national courses were delivered in Tajikistan (n = 28), Uzbekistan (n = 39) and Azerbaijan (n = 30). Participants rated the course highly and demonstrated increased immediate content knowledge after (vs before) course completion (P < .001). CONCLUSIONS: We found that it was feasible to create and deliver a focused critical care short course to clinicians in low‐ and middle‐income countries. Collaboration between WHO, clinical experts, instructional designers, Ministries of Health and local clinician‐leaders facilitated course delivery. Future work should assess its impact on longer‐term knowledge retention and on processes and outcomes of care.


title: Programmed cell removal by calreticulin in tissue homeostasis and cancer

text: Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to “labeling” by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.


title: Where backyard poultry raisers seek care for sick poultry: implications for avian influenza prevention in Bangladesh

text: BACKGROUND: In Bangladesh, backyard poultry raisers lack awareness of avian influenza and infrequently follow government recommendations for its prevention. Identifying where poultry raisers seek care for their ill poultry might help the government better plan how to disseminate avian influenza prevention and control recommendations. METHODS: In order to identify where backyard poultry raisers seek care for their ill poultry, we conducted in-depth and informal interviews: 70 with backyard poultry raisers and six with local poultry healthcare providers in two villages, and five with government veterinary professionals at the sub-district and union levels in two districts during June–August 2009. RESULTS: Most (86% [60/70]) raisers sought care for their backyard poultry locally, 14% used home remedies only and none sought care from government veterinary professionals. The local poultry care providers provided advice and medications (n = 6). Four local care providers had shops in the village market where raisers sought healthcare for their poultry and the remaining two visited rural households to provide poultry healthcare services. Five of the six local care providers did not have formal training in veterinary medicine. Local care providers either did not know about avian influenza or considered avian influenza to be a disease common among commercial but not backyard poultry. The government professionals had degrees in veterinary medicine and experience with avian influenza and its prevention. They had their offices at the sub-district or union level and lacked staffing to reach the backyard raisers at the village level. CONCLUSIONS: The local poultry care providers provided front line healthcare to backyard poultry in villages and were a potential source of information for the rural raisers. Integration of these local poultry care providers in the government’s avian influenza control programs is a potentially useful approach to increase poultry raisers’ and local poultry care providers’ awareness about avian influenza.


title: Synergistic Activity of Colistin in Combination With Resveratrol Against Colistin-Resistant Gram-Negative Pathogens

text: Objectives: In this study, we investigated the antimicrobial activity of resveratrol in combination with colistin, a last-resort agent for the treatment of severe infections caused by multidrug resistant Gram-negative pathogens. Methods: The synergistic activity and the bactericidal activity of colistin in combination with resveratrol was investigated by checkerboard assays and time-kill assays, respectively. A total of 21 strains were investigated, including 16 strains of different species (Klebsiella pneumoniae, n = 6, Escherichia coli, n = 6; Citrobacter braakii, n = 1; Stenotrophomonas malthophilia, n = 1; Enterobacter cloaceae, n = 1; Acinetobacter baumannii, n = 1) with acquired colistin resistance, three colistin-susceptible K. pneumoniae precursors, and two strains of intrinsically colistin-resistant species (Serratia marcescens, n = 1; Proteus mirabilis, n = 1). Mechanisms of acquired colistin resistance included chromosomal mutations (i.e., mgrB, pmrAB) and plasmid genes (mcr-1, mcr-1.2). Results: Resveratrol did not show any significant intrinsic antimicrobial activity. Overall, a relevant synergistic antimicrobial activity of resveratrol in combination with colistin was observed with all tested strains, except for the three colistin-susceptible K. pneumoniae strains, and for two mcr-1-positive E. coli strains. In time-kill assays, performed with 15 selected strains, the combination of colistin 2 mg/L plus resveratrol 128 mg/L was bactericidal with 11 strains, and bacteriostatic for the remaining ones. Conclusions: Resveratrol was found to potentiate colistin activity against a wide panel of colistin-resistant strains, regardless of species and resistance mechanisms, which would deserve further investigation for potential clinical applications.


title: Identification and Validation of Reference Genes for RT-qPCR Normalization in Mythimna separata (Lepidoptera: Noctuidae)

text: Mythimna separata is a major agricultural pest with seasonal migrating trait in China. Formation and regulation mechanism of migration behavior has resulted in a large number of fundamental researches involving quantitative studies of gene expression in this species. Using appropriate reference gene is critical in RT-qPCR data normalization. A comprehensive study on the reference genes in M. separata is lacking. In this paper, expression stabilities of ten candidate reference genes were evaluated in M. separata under various biotic and abiotic conditions by employing four different software geNorm, NormFinder, BestKeeper, and the comparative ΔCT method. The comprehensive stabilities ranking of these genes were suggested by RefFinder. PKG as a target gene was employed to justify the number of reference genes in four larval tissues and two photoperiod treatments. Results demonstrate that the first three most stable genes were as follows: EF, CypA, and β-TUB for developmental stages; EF, CypA, and RPL12 for larval tissues; EF, TBP, and β-TUB for adult tissues. RPL12, β-TUB, and EF for densities; EF, RPL12, and GAPDH for photoperiod treatments; β-TUB, EF, and ATPase for temperature treatments. Stable reference gene combinations may reduce bias in normalization. This work provides for the first time a comprehensive list of appropriate reference genes and facilitates future studies on gene function of M. separata.


title: The impact of respiratory viruses on lung health after preterm birth

text: Children born preterm, less than 37 weeks’ gestation, are at increased risk of viral respiratory infections and associated complications both during their initial birth hospitalisation and in their first years following discharge. This increased burden of viral respiratory infections is likely to have long term implications for lung health and function in individuals born preterm, particularly those with bronchopulmonary dysplasia. Several hypotheses have been put forward to explain the association between early life viral respiratory infection and development of suboptimal lung health and function later in life following preterm birth. Although preterm infants with diminished lung function, particularly small airways, might be particularly susceptible to asthma and wheezing disorders following viral infection, there is evidence that respiratory viruses can activate number of inflammatory and airway re-modelling pathways. Therefore, the aim of this review is to highlight the perinatal and early life risk factors that may contribute to increased susceptibility to viral respiratory infections among preterm infants during early life and to understand how respiratory viral infection may influence the development of abnormal lung health and function later in life.


title: Advances in Designing and Developing Vaccines, Drugs, and Therapies to Counter Ebola Virus

text: Ebola virus (EBOV), a member of the family Filoviridae, is responsible for causing Ebola virus disease (EVD) (formerly named Ebola hemorrhagic fever). This is a severe, often fatal illness with mortality rates varying from 50 to 90% in humans. Although the virus and associated disease has been recognized since 1976, it was only when the recent outbreak of EBOV in 2014–2016 highlighted the danger and global impact of this virus, necessitating the need for coming up with the effective vaccines and drugs to counter its pandemic threat. Albeit no commercial vaccine is available so far against EBOV, a few vaccine candidates are under evaluation and clinical trials to assess their prophylactic efficacy. These include recombinant viral vector (recombinant vesicular stomatitis virus vector, chimpanzee adenovirus type 3-vector, and modified vaccinia Ankara virus), Ebola virus-like particles, virus-like replicon particles, DNA, and plant-based vaccines. Due to improvement in the field of genomics and proteomics, epitope-targeted vaccines have gained top priority. Correspondingly, several therapies have also been developed, including immunoglobulins against specific viral structures small cell-penetrating antibody fragments that target intracellular EBOV proteins. Small interfering RNAs and oligomer-mediated inhibition have also been verified for EVD treatment. Other treatment options include viral entry inhibitors, transfusion of convalescent blood/serum, neutralizing antibodies, and gene expression inhibitors. Repurposed drugs, which have proven safety profiles, can be adapted after high-throughput screening for efficacy and potency for EVD treatment. Herbal and other natural products are also being explored for EVD treatment. Further studies to better understand the pathogenesis and antigenic structures of the virus can help in developing an effective vaccine and identifying appropriate antiviral targets. This review presents the recent advances in designing and developing vaccines, drugs, and therapies to counter the EBOV threat.


title: Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms

text: Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain’s immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00401-018-1885-0) contains supplementary material, which is available to authorized users.


title: Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes

text: The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37–40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe–template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.


title: Oral administration of inactivated porcine epidemic diarrhea virus activate DCs in porcine Peyer’s patches

text: BACKGROUND: Peyer’s patches (PPs) can be considered as the immune site of the intestine. Within PPs, Dendritic cells (DCs) can uptake antigens from the gut lumen by extending dendrites into epithelium, and process it and then present to lymphocytes, which effectively antigen produces an immune response. Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), an acute and highly contagious enteric viral disease. The interaction between inactivated porcine epidemic diarrhea virus and porcine monocyte-derived dendritic cells (Mo-DCs) has been reported. However, little is known about the interaction between inactivated PEDV and DCs in porcine PPs. RESULTS: In this study, for the first time we investigated the role of DCs in porcine PPs after oral administration inactivated PEDV. Firstly, a method to isolate DCs from porcine PPs was established, in which the purity of SWC3a(+)/MHC-II(+) DCs was more than 90%. Our findings clearly indicate that DCs in porcine PPs after oral administration of inactivated PEDV not only stimulated the proliferation of allogeneic lymphocytes, but also secreted cytokines (IL-1, IL-4). Furthermore, the number of DCs and IgA(+) cells in porcine intestinal mucosal significantly increased and the levels of anti-PEDV specific IgG antibody in the serum and SIgA antibody in the feces increased after oral administration inactivated PEDV. CONCLUSIONS: Our findings indicate that oral administration of inactivated PEDV activate DCs in porcine Peyer’s patches and inactivated PEDV may be a useful and safe vaccine to trigger adaptive immunity.


title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production

text: Infectious HCV carrying reporter genes have further applications in understanding the HCV life cycle including replication, viral assembly and release. In this study, a full-length 3039bp LacZ gene was inserted into the derivative of JFH1-AM120 to develop an additional reporter virus. The results showed that the recombinant reporter virus JFH1-AM120-LacZ can replicate and produce lower titers of infectious virus. However, insertion of the LacZ gene in the C-terminal region of the NS5A in HCV JFH1-AM120-LacZ decreased viral replication and dramatically impaired the production of infectious viral particles. Moreover, the JFH1-AM120-LacZ reporter virus lost the LacZ gene after serial passage. Nevertheless, the JFH1-AM120-LacZ reporter virus displayed the entire life cycle of HCV, from replication to production of infectious virus, in Huh7.5 cells. This study demonstrates that the NS5A region of HCV JFH1-AM120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site.


title: Combining New Non-Nucleoside Reverse Transcriptase Inhibitors (RTIs) with AZT Results in Strong Synergism against Multi-RTI-Resistant HIV-1 Strains

text: Reverse transcriptase inhibitors (RTIs), including nucleoside RTIs (NRTIs) and non-nucleoside RTIs (NNRTIs), are critical antiretroviral drugs for the treatment of human immunodeficiency virus (HIV) infection. Emergence of multi-RTI resistance calls for the development of more potent therapeutics or regimens against RTI-resistant strains. Here, we demonstrated that combining azidothymidine (AZT) with a new NNRTIs under development, diarylpyridine (DAPA)-2e, diarylanilin (DAAN)-14h, or DAAN-15h, resulted in strong synergism against infection by divergent HIV-1 strains, including those resistant to NRTIs and NNRTIs, suggesting the potential for developing these novel NNRTIs as salvage therapy for HIV/acquired immune deficiency syndrome (AIDS) patients.


title: Recent Reports of Solid-Phase Cyclohexapeptide Synthesis and Applications

text: Macrocyclic peptides are privileged scaffolds for drug development and constitute a significant portion of macrocyclic drugs on the market today in fields spanning from infectious disease to oncology. Developing orally bioavailable peptide-based drugs remains a challenging task; however, macrocyclization of linear peptides can be an effective strategy to improve membrane permeability, proteolytic stability, oral bioavailability, and overall drug-like characteristics for this class. Significant advances in solid-phase peptide synthesis (SPPS) have enabled the efficient construction of macrocyclic peptide and peptidomimetic libraries with macrolactamization being performed on-resin or in solution phase. The primary goal of this review is to summarize solid-phase cyclohexapeptide synthesis using the on-resin and solution-phase macrocyclization methodologies published since 2013. We also highlight their broad applications ranging from natural product total synthesis, synthetic methodology development, and medicinal chemistry, to drug development and analyses of conformational and physiochemical properties.


title: Generating genomic platforms to study Candida albicans pathogenesis

text: The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein–protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.


title: Deficient humoral responses and disrupted B-cell immunity are associated with fatal SFTSV infection

text: Severe Fever with Thrombocytopenia Syndrome (SFTS), an emerging infectious disease caused by a novel phlebovirus, is associated with high fatality. Therapeutic interventions are lacking and disease pathogenesis is yet to be fully elucidated. The anti-viral immune response has been reported, but humoral involvement in viral pathogenesis is poorly understood. Here we show defective serological responses to SFTSV is associated with disease fatality and a combination of B-cell and T-cell impairment contribute to disruption of anti-viral immunity. The serological profile in deceased patients is characterized by absence of specific IgG to viral nucleocapsid and glycoprotein due to failure of B-cell class switching. Expansion and impairment of antibody secretion is a signature of fatal SFTSV infection. Apoptosis of monocytes in the early stage of infection diminishes antigen-presentation by dendritic cells, impedes differentiation and function of T follicular helper cells, and contributes to failure of the virus-specific humoral response.


title: Aspergillus PCR in Bronchoalveolar Lavage Fluid for the Diagnosis and Prognosis of Aspergillosis in Patients With Hematological and Non-hematological Conditions

text: Objectives: We evaluated the usefulness of an Aspergillus fumigatus quantitative PCR assay performed in bronchoalveolar lavage fluid (BAL) for the diagnosis and prognosis of both invasive and non-invasive aspergillosis. Methods: This 4-year retrospective study involved 613 at-risk patients who had either hematological disorders or other immunosuppressive conditions, notably solid organ transplants. Thirty-five patients had proven/probable aspergillosis and thirteen had chronic non-invasive aspergillosis. We compared PCR, galactomannan index and mycological analysis of BAL. Results: For invasive aspergillosis (IA), PCR performed in BAL yielded 88.6% sensitivity and 95.5% specificity. Comparatively, galactomannan index and mycological examination yielded only 56.3 and 63.6% sensitivity and 97.6 and 94.5% specificity, respectively. Considering the 13 chronic aspergillosis cases, PCR, galactomannan index and mycological examination yielded 76.9, 15.4, and 84.6% sensitivity and 92.2, 94.9, and 93% specificity, respectively. Fungal load in BAL evaluated by PCR was able to discriminate between aspergillosis and contamination, but not between invasive and non-invasive forms. Finally, fungal load was predictive of 90-day mortality, with 23.1% mortality for patients with less than 500 copies/mL versus 68.4% for patients above that cut-off (p < 0.05). Conclusion: Our results indicate that Aspergillus PCR in BAL is of particular interest for both the diagnosis and the prognosis of IA. It is likewise an interesting tool for the diagnosis of non-invasive forms.


title: Immunological memory cells

text: This article reviews immunological memory cells, currently represented by T and B lymphocytes and natural killer (NK) cells, which determine a rapid and effective response against a second encounter with the same antigen. Among T lymphocytes, functions of memory cells are provided by their subsets: central memory, effector memory, tissue-resident memory, regulatory memory and stem memory T cells. Memory T and B lymphocytes have an essential role in the immunity against microbial pathogens but are also involved in autoimmunity and maternal-fetal tolerance. Furthermore, the evidence of immunological memory has been established for NK cells. NK cells can respond to haptens or viruses, which results in generation of antigen-specific memory cells. T, B and NK cells, which have a role in immunological memory, have been characterized phenotypically and functionally. During the secondary immune response, these cells are involved in the reaction against foreign antigens, including pathogens, and take part in autoimmune diseases, but also are crucial to immunological tolerance and vaccine therapy.


docid: pxvrqxxf

title: Potential and challenges of tannins as an alternative to in-feed antibiotics for farm animal production

text: Naturally occurring plant compounds including tannins, saponins and essential oils are extensively assessed as natural alternatives to in-feed antibiotics. Tannins are a group of polyphenolic compounds that are widely present in plant region and possess various biological activities including antimicrobial, anti-parasitic, anti-viral, antioxidant, anti-inflammatory, immunomodulation, etc. Therefore, tannins are the major research subject in developing natural alternative to in-feed antibiotics. Strong protein affinity is the well-recognized property of plant tannins, which has successfully been applied to ruminant nutrition to decrease protein degradation in the rumen, and thereby improve protein utilization and animal production efficiency. Incorporations of tannin-containing forage in ruminant diets to control animal pasture bloat, intestinal parasite and pathogenic bacteria load are another 3 important applications of tannins in ruminant animals. Tannins have traditionally been regarded as “anti-nutritional factor” for monogastric animals and poultry, but recent researches have revealed some of them, when applied in appropriate manner, improved intestinal microbial ecosystem, enhanced gut health and hence increased productive performance. The applicability of plant tannins as an alternative to in-feed antibiotics depends on many factors that contribute to the great variability in their observed efficacies.


title: Isolation and characterization of novel bat paramyxovirus B16-40 potentially belonging to the proposed genus Shaanvirus

text: The bat paramyxovirus B16-40 was first isolated in Korea in this study. Using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. In the phylogenetic analysis, the virus was found to belong to the recently proposed genus Shaanvirus. Through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (HN) protein as one of the structural proteins. When mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus 1 in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus 1. In addition, the bat paramyxovirus B16-40 was not infectious in the mouse model. Collectively, this study provided basic information on further classification of the bat paramyxovirus B16-40 and related viruses in the proposed genus Shaanvirus.


title: Why are RNA virus mutation rates so damn high?

text: The high mutation rate of RNA viruses is credited with their evolvability and virulence. This Primer, however, discusses recent evidence that this is, in part, a byproduct of selection for faster genomic replication.


title: Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice

text: During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR(-/-) mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.


title: Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces

text: Interactions of influenza A virus (IAV) with sialic acid (SIA) receptors determine viral fitness and host tropism. Binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (HA), a receptor-destroying neuraminidase (NA) and a complex in vivo receptor-repertoire. The crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. In this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of IAV binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of NA activity. In addition to HA, NA was shown to be able to contribute to the initial binding rate while catalytically active. Virus-receptor binding in turn contributed to receptor cleavage by NA. Multiple low-affinity HA-SIA interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which NA activity was driving rolling of virus particles over the receptor-surface. Virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to NA activity of rolling IAV particles. The results indicate that in vivo IAV particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the HA-NA-receptor balance. Quantitative BLI analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by IAV but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein.


title: Prevalence of Respiratory Polyomaviruses Among Pediatric Patients With Respiratory Symptoms in Singapore

text: Background: Although WU polyomavirus (WU) and KI polyomavirus (KI) have been demonstrated to infect the human respiratory tract, it remains unclear if WU or KI cause human disease. We sought to further investigate the relationship between WU and KI infection and respiratory disease in a pediatric population with respiratory symptoms in Singapore. Methods: We conducted a cross-sectional study of pediatric patients with respiratory symptoms in a Singaporean pediatrics hospital. Upon consent, residual respiratory samples from pediatric inpatients, previously screened for common respiratory viruses, were collected and further screened for WU and KI using qPCR. The amplicons of positive samples were sequenced for confirmation. The severity of a patient's illness was assessed by chart review post-discharge looking for clinical markers of respiratory status such as presenting symptoms, diagnoses, and interventions. Results: From December 2016 to April 2017, 201 patients with residual respiratory samples were enrolled in the study. The average age of all participants recruited was 45 months. WU and KI were detected in 13% (26/201) and 3% (6/201) of patients, respectively. Conducting bivariate and multivariate modeling, patients with WU or KI positivity were not at increased risk of SARI, need for additional oxygen, intravenous fluids, and did not receive additional oral antibiotics or bronchodilators during admission. In contrast, patients with RSV detections were at increased risk of requiring supplemental oxygen during hospital admission. Conclusion: While limited in sample size, our pilot study data do not support the hypothesis that molecular evidence of WU or KI was associated with increased morbidity among a sample of general, pediatric patients with respiratory illness in Singapore.


title: Population Serologic Immunity to Human and Avian H2N2 Viruses in the United States and Hong Kong for Pandemic Risk Assessment

text: BACKGROUND: Influenza A pandemics cause significant mortality and morbidity. H2N2 viruses have caused a prior pandemic, and are circulating in avian reservoirs. The age-related frequency of current population immunity to H2 viruses was evaluated. METHODS: Hemagglutinin inhibition (HAI) assays against historical human and recent avian influenza A(H2N2) viruses were performed across age groups in Rochester, New York, and Hong Kong, China. The impact of existing cross-reactive HAI immunity on the effective reproduction number was modeled. RESULTS: One hundred fifty individual sera from Rochester and 295 from Hong Kong were included. Eighty-five percent of patients born in Rochester and Hong Kong before 1968 had HAI titers ≥1:40 against A/Singapore/1/57, and >50% had titers ≥1:40 against A/Berkeley/1/68. The frequency of titers ≥1:40 to avian H2N2 A/mallard/England/727/06 and A/mallard/Netherlands/14/07 in subjects born before 1957 was 62% and 24%, respectively. There were no H2 HAI titers >1:40 in individuals born after 1968. These levels of seroprevalence reduce the initial reproduction number of A/Singapore/1/1957 or A/Berkeley/1/68 by 15%–20%. A basic reproduction number (R(0)) of the emerging transmissible virus <1.2 predicts a preventable pandemic. CONCLUSIONS: Population immunity to H2 viruses is insufficient to block epidemic spread of H2 virus. An H2N2 pandemic would have lower impact in those born before 1968.


title: Accounting for non-stationarity in epidemiology by embedding time-varying parameters in stochastic models

text: The spread of disease through human populations is complex. The characteristics of disease propagation evolve with time, as a result of a multitude of environmental and anthropic factors, this non-stationarity is a key factor in this huge complexity. In the absence of appropriate external data sources, to correctly describe the disease propagation, we explore a flexible approach, based on stochastic models for the disease dynamics, and on diffusion processes for the parameter dynamics. Using such a diffusion process has the advantage of not requiring a specific mathematical function for the parameter dynamics. Coupled with particle MCMC, this approach allows us to reconstruct the time evolution of some key parameters (average transmission rate for instance). Thus, by capturing the time-varying nature of the different mechanisms involved in disease propagation, the epidemic can be described. Firstly we demonstrate the efficiency of this methodology on a toy model, where the parameters and the observation process are known. Applied then to real datasets, our methodology is able, based solely on simple stochastic models, to reconstruct complex epidemics, such as flu or dengue, over long time periods. Hence we demonstrate that time-varying parameters can improve the accuracy of model performances, and we suggest that our methodology can be used as a first step towards a better understanding of a complex epidemic, in situation where data is limited and/or uncertain.


title: Estimating the Asymptomatic Ratio of Norovirus Infection During Foodborne Outbreaks With Laboratory Testing in Japan

text: BACKGROUND: Foodborne norovirus outbreak data in Japan from 2005–2006, involving virological surveillance of all symptomatic and asymptomatic individuals, were reanalyzed to estimate the asymptomatic ratio of norovirus infection along with the risk of infection and the probability of virus shedding. METHODS: Employing a statistical model that is considered to capture the data-generating process of the outbreak and virus surveillance, maximum likelihood estimation of the asymptomatic ratio was implemented. RESULTS: Assuming that all norovirus outbreaks (n = 55) were the result of random sampling from an identical distribution and ignoring genogroup and genotype specificities, the asymptomatic ratio was estimated at 32.1% (95% confidence interval [CI], 27.7–36.7). Although not significant, separate estimation of the asymptomatic ratio of the GII.4 genotype appeared to be greater than other genotypes and was estimated at 40.7% (95% CI, 32.8–49.0). CONCLUSION: The present study offered the first explicit empirical estimates of the asymptomatic ratio of norovirus infection in natural infection settings. The estimate of about 30% was consistent with those derived from volunteer challenge studies. Practical difficulty in controlling GII.4 outbreaks was supported by the data, considering that a large estimate of the asymptomatic ratio was obtained for the GII.4 genotype.