Modeling the CD8＋ T cell immune response to influenza infection in adult and aged mice.

The CD8＋ T cell response is the main determinant of viral clearance during influenza infection. However, influenza viral dynamics and the respective immune responses are affected by the host's age. To investigate age-related differences in the CD8＋ T cell immune response dynamics, we propose an 16 ordinary differential equation models of existing experimental data. These data consist of viral titer and CD8＋ T cell counts collected periodically over a period of 19 days from adult and aged mice infected with influenza A/Puerto Rico/8/34 (H1N1). We use the corrected Akaike Information Criterion to identify the models which best represent the considered data. Our model selection process indicates differences in mechanisms which reduce the CD8＋ T cell response: linear downregulation is favored for adult mice, while baseline exponential decay is favored for aged mice. Parameter fitting of the top ranked models suggests that the aged population has reduced CD8＋ T cell proliferation compared to the adult population. More experimental work is needed to determine the specific immunological features through which age might cause these differences. A better understanding of the immunological mechanisms by which aging leads to discrepant CD8＋ T cell dynamics may inform future treatment strategies.

Extracellular ISG15 triggers ISGylation via a type-I interferon independent mechanism to regulate host response during virus infection.

Type-I interferons (IFN) are key antiviral factors that induce several cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity. Apart from ISGylation, unconjugated ISG15 is also released from cells during immuno-stimulation and virus infection. The role of extracellular ISG15 during virus infection was unknown. We now show that extracellular ISG15 acts as a soluble antiviral factor to restrict virus infection in human epithelial cells via an IFN-independent mechanism. Additionally, we demonstrated that extracellular ISG15 induces expression of intracellular ISG15 and ISGylation in the absence of IFN. Mechanistic studies revealed that extracellular ISG15 interacts with cell surface integrin (α5ß1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified ISG15 protein as a new soluble factor that confers IFN-independent antiviral activity by inducing ISGylation. Additionally, we have unfolded an IFN-independent non-canonical ISGylation by extracellular ISG15 that operates via the integrin-FAK pathway.

Human beta defensin-3 mediated activation of ß-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5.

Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects the respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccines to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Cytoplasmic stabilization of ß-catenin protein results in activation of canonical Wingless (Wnt)/ß-catenin signaling pathway that culminates in transcriptional activation of various genes regulated by T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This pathway is involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering ß-catenin protein stabilization and induction of ß-catenin mediated transcriptional activity. Functionally, the activated ß-catenin pathway promoted a pro-inflammatory response during RSV infection of lung epithelial cells. Studies with ß-catenin inhibitors and A549 cells lacking optimal ß-catenin activity demonstrated a significant loss of pro-inflammatory chemokine interleukin-8 (IL-8) release from RSV-infected cells. Mechanistically, our studies revealed a role of extracellular human beta defensin-3 (HBD3) in interacting with cell surface Wnt receptor LDL receptor-related protein-5 (LRP5) to activate the non-canonical Wnt independent ß-catenin pathway during RSV infection. We showed gene expression and release of HBD3 from RSV-infected cells and silencing of HBD3 expression resulted in reduced stabilization of ß-catenin protein during RSV infection. Furthermore, we observed the binding of extracellular HBD3 with cell surface localized LRP5 protein, and our in silico and protein-protein interaction studies have highlighted a direct interaction of HBD3 with LRP5. Thus, our studies have identified the ß-catenin pathway as a key regulator of pro-inflammatory response during RSV infection of human lung epithelial cells. This pathway was induced during RSV infection via a non-canonical Wnt-independent mechanism involving paracrine/autocrine action of extracellular HBD3 activating cell surface Wnt receptor complex by directly interacting with the LRP5 receptor.

Proinflammatory Responses in SARS-CoV-2 and Soluble Spike Glycoprotein S1 Subunit Activated Human Macrophages.

Critically ill COVID-19 patients display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We inoculated and treated human macrophage cell line THP-1 with SARS-CoV-2 and purified, glycosylated, soluble SARS-CoV-2 spike protein S1 subunit (S1) to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication or viral entry, virus exposure resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that extracellular soluble S1 protein is a key viral component inducing pro-inflammatory responses in macrophages, independent of virus replication. Thus, virus- or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.

Hidden variable models reveal the effects of infection from changes in host survival.

The impacts of disease on host vital rates can be demonstrated using longitudinal studies, but these studies can be expensive and logistically challenging. We examined the utility of hidden variable models to infer the individual effects of infectious disease from population-level measurements of survival when longitudinal studies are not possible. Our approach seeks to explain temporal deviations in population-level survival after introducing a disease causative agent when disease prevalence cannot be directly measured by coupling survival and epidemiological models. We tested this approach using an experimental host system (Drosophila melanogaster) with multiple distinct pathogens to validate the ability of the hidden variable model to infer per-capita disease rates. We then applied the approach to a disease outbreak in harbor seals (Phoca vituline) that had data on observed strandings but no epidemiological data. We found that our hidden variable modeling approach could successfully detect the per-capita effects of disease from monitored survival rates in both the experimental and wild populations. Our approach may prove useful for detecting epidemics from public health data in regions where standard surveillance techniques are not available and in the study of epidemics in wildlife populations, where longitudinal studies can be especially difficult to implement.

Kathryn V. Holmes: A Career of Contributions to the Coronavirus Field.

Over the past two years, scientific research has moved at an unprecedented rate in response to the COVID-19 pandemic. The rapid development of effective vaccines and therapeutics would not have been possible without extensive background knowledge on coronaviruses developed over decades by researchers, including Kathryn (Kay) Holmes. Kay's research team discovered the first coronavirus receptors for mouse hepatitis virus and human coronavirus 229E and contributed a wealth of information on coronaviral spike glycoproteins and receptor interactions that are critical determinants of host and tissue specificity. She collaborated with several research laboratories to contribute knowledge in additional areas, including coronaviral pathogenesis, epidemiology, and evolution. Throughout her career, Kay was an extremely dedicated and thoughtful mentor to numerous graduate students and post-doctoral fellows. This article provides a review of her contributions to the coronavirus field and her exemplary mentoring.

Molecular Modeling Predicts Novel Antibody Escape Mutations in the Respiratory Syncytial Virus Fusion Glycoprotein.

Monoclonal antibodies are increasingly used for the prevention and/or treatment of viral infections. One caveat of their use is the ability of viruses to evolve resistance to antibody binding and neutralization. Computational strategies to identify viral mutations that may disrupt antibody binding would leverage the wealth of viral genomic sequence data to monitor for potential antibody-resistant mutations. The respiratory syncytial virus is an important pathogen for which monoclonal antibodies against the fusion (F) protein are used to prevent severe disease in high-risk infants. In this study, we used an approach that combines molecular dynamics simulations with FoldX to estimate changes in free energy in F protein folding and binding to the motavizumab antibody upon each possible amino acid change. We systematically selected 8 predicted escape mutations and tested them in an infectious clone. Consistent with our F protein stability predictions, replication-effective viruses were observed for each selected mutation. Six of the eight variants showed increased resistance to neutralization by motavizumab. Flow cytometry was used to validate the estimated (model-predicted) effects on antibody binding to F. Using surface plasmon resonance, we determined that changes in the on-rate of motavizumab binding were associated with the reduced affinity for two novel escape mutations. Our study empirically validated the accuracy of our molecular modeling approach and emphasized the role of biophysical protein modeling in predicting viral resistance to antibody-based therapeutics that can be used to monitor the emergence of resistant viruses and to design improved therapeutic antibodies. IMPORTANCE Respiratory syncytial virus (RSV) causes severe disease in young infants, particularly those with heart or lung diseases or born prematurely. Because no vaccine is currently available, monoclonal antibodies are used to prevent severe RSV disease in high-risk infants. While it is known that RSV evolves to avoid recognition by antibodies, screening tools that can predict which changes to the virus may lead to antibody resistance are greatly needed.

Priming With Rhinovirus Protects Mice Against a Lethal Pulmonary Coronavirus Infection.

Rhinoviruses (RV) have been shown to inhibit subsequent infection by heterologous respiratory viruses, including influenza viruses and severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). To better understand the mechanisms whereby RV protects against pulmonary coronavirus infection, we used a native murine virus, mouse hepatitis virus strain 1 (MHV-1), that causes severe disease in the lungs of infected mice. We found that priming of the respiratory tract with RV completely prevented mortality and reduced morbidity of a lethal MHV-1 infection. Replication of MHV-1 was reduced in RV-primed mouse lungs although expression of antiviral type I interferon, IFN-ß, was more robust in mice infected with MHV-1 alone. We further showed that signaling through the type I interferon receptor was required for survival of mice given a non-lethal dose of MHV-1. RV-primed mice had reduced pulmonary inflammation and hemorrhage and influx of leukocytes, especially neutrophils, in the airways upon MHV-1 infection. Although MHV-1 replication was reduced in RV-primed mice, RV did not inhibit MHV-1 replication in coinfected lung epithelial cells in vitro. In summary, RV-mediated priming in the respiratory tract reduces viral replication, inflammation, and tissue damage, and prevents mortality of a pulmonary coronavirus infection in mice. These results contribute to our understanding of how distinct respiratory viruses interact with the host to affect disease pathogenesis, which is a critical step in understanding how respiratory viral coinfections impact human health.

Proinflammatory responses in SARS-CoV-2 infected and soluble spike glycoprotein S1 subunit activated human macrophages.

Critically ill COVID-19 patients infected with SARS-CoV-2 display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We used SARS-CoV-2 infected and glycosylated soluble SARS-CoV-2 Spike S1 subunit (S1) treated THP-1 human-derived macrophage-like cell line to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication, virus infection resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that S1 is a key viral component inducing inflammatory response in macrophages, independently of virus replication. Thus, virus-infected or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.

Rhinovirus Reduces the Severity of Subsequent Respiratory Viral Infections by Interferon-Dependent and -Independent Mechanisms.

Coinfection by heterologous viruses in the respiratory tract is common and can alter disease severity compared to infection by individual virus strains. We previously found that inoculation of mice with rhinovirus (RV) 2 days before inoculation with a lethal dose of influenza A virus [A/Puerto Rico/8/34 (H1N1) (PR8)] provides complete protection against mortality. Here, we extended that finding to a second lethal respiratory virus, pneumonia virus of mice (PVM), and analyzed potential mechanisms of RV-induced protection. RV completely prevented mortality and weight loss associated with PVM infection. Major changes in host gene expression upon PVM infection were delayed compared to PR8. RV induced earlier recruitment of inflammatory cells, which were reduced at later times in RV-inoculated mice. Findings common to both virus pairs included the upregulated expression of mucin-associated genes and dampening of inflammation-related genes in mice that were inoculated with RV before lethal virus infection. However, type I interferon (IFN) signaling was required for RV-mediated protection against PR8 but not PVM. IFN signaling had minor effects on PR8 replication and contributed to controlling neutrophilic inflammation and hemorrhagic lung pathology in RV/PR8-infected mice. These findings, combined with differences in virus replication levels and disease severity, suggest that the suppression of inflammation in RV/PVM-infected mice may be due to early, IFN-independent suppression of viral replication, while that in RV/PR8-infected mice may be due to IFN-dependent modulation of immune responses. Thus, a mild upper respiratory viral infection can reduce the severity of a subsequent severe viral infection in the lungs through virus-dependent mechanisms. IMPORTANCE Respiratory viruses from diverse families cocirculate in human populations and are frequently detected within the same host. Although clinical studies suggest that infection by multiple different respiratory viruses may alter disease severity, animal models in which we can control the doses, timing, and strains of coinfecting viruses are critical to understanding how coinfection affects disease severity. Here, we compared gene expression and immune cell recruitment between two pairs of viruses (RV/PR8 and RV/PVM) inoculated sequentially in mice, both of which result in reduced severity compared to lethal infection by PR8 or PVM alone. Reduced disease severity was associated with suppression of inflammatory responses in the lungs. However, differences in disease kinetics and host and viral gene expression suggest that protection by coinfection with RV may be due to distinct molecular mechanisms. Indeed, we found that antiviral cytokine signaling was required for RV-mediated protection against lethal infection by PR8 but not PVM.

Lytic Cell Death Mechanisms in Human Respiratory Syncytial Virus-Infected Macrophages: Roles of Pyroptosis and Necroptosis.

Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages.

Respiratory epithelial cells as master communicators during viral infections.

PURPOSE OF REVIEW: Communication by epithelial cells during respiratory viral infections is critical in orchestrating effective anti-viral responses but also can lead to excessive inflammation. This review will evaluate studies that investigate how respiratory epithelial cells influence the behavior of immune cells and how epithelial cell/immune cell interactions contribute to antiviral responses and immunopathology outcomes. RECENT FINDINGS: Previous studies have characterized cytokine responses of virus-infected epithelial cells. More recent studies have carefully demonstrated the effects of these cytokines on cellular behaviors within the infected lung. Infected epithelial cells release exosomes that specifically regulate responses of monocytes and neighboring epithelial cells without promoting spread of virus. In contrast, rhinovirus-infected cells induce monocytes to upregulate expression of the viral receptor, promoting spread of the virus to alternate cell types. The precise alteration of PDL expression on infected epithelial cells has been shown to switch between inhibition and activation of antiviral responses. SUMMARY: These studies have more precisely defined the interactions between epithelial and immune cells during viral infections. This level of understanding is critical for the development of novel therapeutic strategies that promote effective antiviral responses or epithelial repair, or inhibit damaging inflammatory responses during severe respiratory viral infections.

A two-stage experimental design for dilution assays.

Dilution assays to determine solute concentration have found wide use in biomedical research. Many dilution assays return imprecise concentration estimates because they are only done to orders of magnitude. Previous statistical work has focused on how to design efficient experiments that can return more precise estimates, however this work has not considered the practical difficulties of implementing these designs in the laboratory. We developed a two-stage experiment with a first stage that obtains an order of magnitude estimate and a second stage that concentrates effort on the most informative dilution to increase estimator precision. We show using simulations and an empirical example that the best two-stage experimental designs yield estimates that are remarkably more accurate than standard methods with equivalent effort. This work demonstrates how to utilize previous advances in experimental design in a manner consistent with current laboratory practice. We expect that multi-stage designs will prove to be useful for obtaining precise estimates with minimal experimental effort.

Attenuation of Influenza A Virus Disease Severity by Viral Coinfection in a Mouse Model.

Influenza viruses and rhinoviruses are responsible for a large number of acute respiratory viral infections in human populations and are detected as copathogens within hosts. Clinical and epidemiological studies suggest that coinfection by rhinovirus and influenza virus may reduce disease severity and that they may also interfere with each other's spread within a host population. To determine how coinfection by these two unrelated respiratory viruses affects pathogenesis, we established a mouse model using a minor serogroup rhinovirus (rhinovirus strain 1B [RV1B]) and mouse-adapted influenza A virus (A/Puerto Rico/8/1934 [PR8]). Infection of mice with RV1B 2 days before PR8 reduced the severity of infection by a low or medium, but not high, dose of PR8. Disease attenuation was associated with an early inflammatory response in the lungs and enhanced clearance of PR8. However, coinfection by RV1B did not reduce PR8 viral loads early in infection or inhibit replication of PR8 within respiratory epithelia or in vitro Inflammation in coinfected mice remained focal compared to diffuse inflammation and damage in the lungs of mice infected by PR8. The timing of RV1B coinfection was a critical determinant of protection, suggesting that sufficient time is needed to induce this response. Finally, disease attenuation was not unique to RV1B: dose-dependent coinfection by a murine coronavirus (mouse hepatitis virus strain 1 [MHV-1]) also reduced the severity of PR8 infection. Unlike RV1B, coinfection with MHV-1 reduced early PR8 replication, which was associated with upregulation of beta interferon (IFN-ß) expression. This model is critical for understanding the mechanisms responsible for influenza disease attenuation during coinfection by unrelated respiratory viruses.IMPORTANCE Viral infections in the respiratory tract can cause severe disease and are responsible for a majority of pediatric hospitalizations. Molecular diagnostics have revealed that approximately 20% of these patients are infected by more than one unrelated viral pathogen. To understand how viral coinfection affects disease severity, we inoculated mice with a mild viral pathogen (rhinovirus or murine coronavirus), followed 2 days later by a virulent viral pathogen (influenza A virus). This model demonstrated that rhinovirus can reduce the severity of influenza A virus, which corresponded with an early but controlled inflammatory response in the lungs and early clearance of influenza A virus. We further determined the dose and timing parameters that were important for effective disease attenuation and showed that influenza disease is also reduced by coinfection with a murine coronavirus. These findings demonstrate that coinfecting viruses can alter immune responses and pathogenesis in the respiratory tract.

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus.

The low-cost Open qPCR instrument can be used for different tasks in the aptamer selection process: quantification of DNA, cycle course optimization, screening, and final binding characterization. We have selected aptamers against whole Drosophila C virus (DCV) particles and recombinant epidermal growth factor receptor (EGFR). We performed systematic evolution of ligands by exponential enrichment (SELEX) using the Open qPCR to optimize each amplification step. The Open qPCR instrument identified the best aptamer candidate. The Open qPCR has the capacity to perform melt curves, and we used this function to perform thermofluorimetric analysis (TFA) to quantify target-aptamer binding. We confirmed target-aptamer binding using flow cytometry. A sandwich type luminescence bioassay based on our anti-DCV aptamer was sensitive to DCV and did not respond to a related virus, demonstrating that our selected anti-DCV aptamer can be used to specifically detect DCV.

Lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses.

The severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. Disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. Murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. We used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, RV1B), moderate (coronavirus, MHV-1), and severe (influenza A virus, PR8) disease in mice. RV1B infection caused numerous gene expression changes, but the differential effect peaked at 12 hours post-infection. PR8 altered an intermediate number of genes whose expression continued to change through 24 hours. MHV-1 had comparatively few effects on host gene expression. The viruses elicited highly overlapping responses in antiviral genes, though MHV-1 induced a lower type I interferon response than the other two viruses. Signature genes were identified for each virus and included host defense genes for PR8, tissue remodeling genes for RV1B, and transcription factors for MHV-1. Our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract.

New Perspectives on Ebola Virus Evolution.

Since the recent devastating outbreak of Ebola virus disease in western Africa, there has been significant effort to understand the evolution of the deadly virus that caused the outbreak. There has been a considerable investment in sequencing Ebola virus (EBOV) isolates, and the results paint an important picture of how the virus has spread in western Africa. EBOV evolution cannot be understood outside the context of previous outbreaks, however. We have focused this study on the evolution of the EBOV glycoprotein gene (GP) because one of its products, the spike glycoprotein (GP1,2), is central to the host immune response and because it contains a large amount of the phylogenetic signal for this virus. We inferred the maximum likelihood phylogeny of 96 nonredundant GP gene sequences representing each of the outbreaks since 1976 up to the end of 2014. We tested for positive selection and considered the placement of adaptive amino acid substitutions along the phylogeny and within the protein structure of GP1,2. We conclude that: 1) the common practice of rooting the phylogeny of EBOV between the first known outbreak in 1976 and the next outbreak in 1995 provides a misleading view of EBOV evolution that ignores the fact that there is a non-human EBOV host between outbreaks; 2) the N-terminus of GP1 may be constrained from evolving in response to the host immune system by the highly expressed, secreted glycoprotein, which is encoded by the same region of the GP gene; 3) although the mucin-like domain of GP1 is essential for EBOV in vivo, it evolves rapidly without losing its twin functions: providing O-linked glycosylation sites and a flexible surface.

Initiating a watch list for Ebola virus antibody escape mutations.

The 2014 Ebola virus (EBOV) outbreak in West Africa is the largest in recorded history and resulted in over 11,000 deaths. It is essential that strategies for treatment and containment be developed to avoid future epidemics of this magnitude. With the development of vaccines and antibody-based therapies using the envelope glycoprotein (GP) of the 1976 Mayinga strain, one important strategy is to anticipate how the evolution of EBOV might compromise these efforts. In this study we have initiated a watch list of potential antibody escape mutations of EBOV by modeling interactions between GP and the antibody KZ52. The watch list was generated using molecular modeling to estimate stability changes due to mutation. Every possible mutation of GP was considered and the list was generated from those that are predicted to disrupt GP-KZ52 binding but not to disrupt the ability of GP to fold and to form trimers. The resulting watch list contains 34 mutations (one of which has already been seen in humans) at six sites in the GP2 subunit. Should mutations from the watch list appear and spread during an epidemic, it warrants attention as these mutations may reflect an evolutionary response from the virus that could reduce the effectiveness of interventions such as vaccination. However, this watch list is incomplete and emphasizes the need for more experimental structures of EBOV interacting with antibodies in order to expand the watch list to other epitopes. We hope that this work provokes experimental research on evolutionary escape in both Ebola and other viral pathogens.

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus.

Infection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography-mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in PR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell protein profiles will elucidate cellular processes in IAV pathogenesis, which may provide insight into potential therapies to modulate disease severity.

Neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology.

Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMN-depleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.