Generation and Functional Analysis of Defective Viral Genomes during SARS-CoV-2 Infection.

Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from transcriptome sequencing (RNA-seq) data sets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hot spots were identified for DVG recombination, and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single-cell RNA-seq analysis indicated the interferon (IFN) stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the next-generation sequencing (NGS) data set from a published cohort study and observed a significantly higher amount and frequency of DVG in symptomatic patients than those in asymptomatic patients. Finally, we observed exceptionally diverse DVG populations in one immunosuppressive patient up to 140 days after the first positive test of COVID-19, suggesting for the first time an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and into how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. IMPORTANCE Defective viral genomes (DVGs) are generated ubiquitously in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide the potential for them to be used in novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex, and this recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hot spots for nonhomologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide evidence to harness the immunostimulatory potential of DVGs in the development of a vaccine and antivirals for SARS-CoV-2.

Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.

Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility-thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.

Generation and functional analysis of defective viral genomes during SARS-CoV-2 infection.

Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from RNA-seq datasets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hotspots were identified for DVG recombination and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single cell RNA-seq analysis indicated the IFN stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the NGS dataset from a published cohort study and observed significantly higher DVG amount and frequency in symptomatic patients than that in asymptomatic patients. Finally, we observed unusually high DVG frequency in one immunosuppressive patient up to 140 days after admitted to hospital due to COVID-19, first-time suggesting an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. Importance: Defective viral genomes (DVGs) are ubiquitously generated in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide them the potential for novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex and the recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hotspots for non-homologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide the evidence to harness DVGs’ immunostimulatory potential in the development of vaccine and antivirals for SARS-CoV-2.

CX3CR1 Engagement by Respiratory Syncytial Virus Leads to Induction of Nucleolin and Dysregulation of Cilia-related Genes.

Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G-protein. The motif has been indicated as facilitating attachment of the virus to the host initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wildtype G-protein, or a mutant virus containing a CX4C mutation in the G-protein. RNA sequencing was performed on mock and 4-days-post-infected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G-protein. Here we report that RSV infection resulted in significant changes in the levels 766 transcripts. Many nuclear associated proteins were upregulated in the WT group, including nucleolin. Alternatively, cilia-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G-protein to the culture lead to the suppression of cilia-related genes while also inducing nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription decreasing nucleolin induction and lessening the suppression of cilia-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with cilia function.ImportanceRespiratory Syncytial Virus (RSV) has an enormous impact on infants and the elderly including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G-protein and F-protein, have been shown to bind to human CX3CR1and nucleolin, respectively. Here we show that the G-protein induces nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G-protein, CX4C, reverses these transcriptional changes.

Implementing sequence-based antigenic distance calculation into immunological shape space model.

BACKGROUND: In 2009, a novel influenza vaccine was distributed worldwide to combat the H1N1 influenza "swine flu" pandemic. However, antibodies induced by the vaccine display differences in their specificity and cross-reactivity dependent on pre-existing immunity. Here, we present a computational model that can capture the effect of pre-existing immunity on influenza vaccine responses. The model predicts the region of the virus hemagglutinin (HA) protein targeted by antibodies after vaccination as well as the level of cross-reactivity induced by the vaccine. We tested our model by simulating a scenario similar to the 2009 pandemic vaccine and compared the results to antibody binding data obtained from human subjects vaccinated with the monovalent 2009 H1N1 influenza vaccine. RESULTS: We found that both specificity and cross-reactivity of the antibodies induced by the 2009 H1N1 influenza HA protein were affected by the viral strain the individual was originally exposed. Specifically, the level of antigenic relatedness between the original exposure HA antigen and the 2009 HA protein affected antigenic-site immunodominance. Moreover, antibody cross-reactivity was increased when the individual's pre-existing immunity was specific to an HA protein antigenically distinct from the 2009 pandemic strain. Comparison of simulation data with antibody binding data from human serum samples demonstrated qualitative and quantitative similarities between the model and real-life immune responses to the 2009 vaccine. CONCLUSION: We provide a novel method to evaluate expected outcomes in antibody specificity and cross-reactivity after influenza vaccination in individuals with different influenza HA antigen exposure histories. The model produced similar outcomes as what has been previously reported in humans after receiving the 2009 influenza pandemic vaccine. Our results suggest that differences in cross-reactivity after influenza vaccination should be expected in individuals with different exposure histories.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Collagen VI Deficiency Results in Structural Abnormalities in the Mouse Lung.

Collagen VI (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. Lung structure and organization were studied in a previously described Col6a1-/- mouse, which does not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed histopathologic alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%; P < 0.01) and fewer (31%; P < 0.001) alveoli. These airspace abnormalities included reduced isolectin B4+ alveolar capillaries and surfactant protein C-positive alveolar epithelial type-II cells. Alterations in lung function consistent with these histopathologic changes were evident. Col6a1-/- mice also displayed multiple airway changes, including increased branching (59%; P < 0.001), increased mucosal thickness (34%; P < 0.001), and increased epithelial cell density (13%; P < 0.001). Comprehensive transcriptome analysis revealed that the loss of COL6 is associated with reductions in integrin-paxillin-phosphatidylinositol 3-kinase signaling in vivo. In vitro, COL6 promoted steady-state phosphorylated paxillin levels and reduced cell density (16% to 28%; P < 0.05) at confluence. Inhibition of phosphatidylinositol 3-kinase, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung.

BACKGROUND: Data on the host factors that contribute to infection of young children by respiratory syncytial virus (RSV) are limited. The human chemokine receptor, CX3CR1, has recently been implicated as an RSV receptor. Here we evaluate a role for CX3CR1 in pediatric lung RSV infections. METHODS: CX3CR1 transcript levels in the upper and lower pediatric airways were assessed. Tissue localization and cell-specific expression was confirmed using in situ hybridization and immunohistochemistry. The role of CX3CR1 in RSV infection was also investigated using a novel physiological model of pediatric epithelial cells. RESULTS: Low levels of CX3CR1 transcript were often, but not always, expressed in both upper (62%) and lower airways (36%) of pediatric subjects. CX3CR1 transcript and protein expression was detected in epithelial cells of normal human pediatric lung tissues. CX3CR1 expression was readily detected on primary cultures of differentiated pediatric/infant human lung epithelial cells. RSV demonstrated preferential infection of CX3CR1-positive cells, and blocking CX3CR1/RSV interaction significantly decreased viral load. CONCLUSION: CX3CR1 is present in the airways of pediatric subjects where it may serve as a receptor for RSV infection. Furthermore, CX3CR1 appears to play a mechanistic role in mediating viral infection of pediatric airway epithelial cells in vitro.

Programming of Distinct Chemokine-Dependent and -Independent Search Strategies for Th1 and Th2 Cells Optimizes Function at Inflamed Sites.

T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.

Mutation of Respiratory Syncytial Virus G Protein's CX3C Motif Attenuates Infection in Cotton Rats and Primary Human Airway Epithelial Cells.

Despite being a high priority for vaccine development, no vaccine is yet available for respiratory syncytial virus (RSV). A live virus vaccine is the primary type of vaccine being developed for young children. In this report, we describe our studies of infected cotton rats and primary human airway epithelial cells (pHAECs) using an RSV r19F with a mutation in the CX3C chemokine motif in the RSV G protein (CX4C). Through this CX3C motif, RSV binds to the corresponding chemokine receptor, CX3CR1, and this binding contributes to RSV infection of pHAECs and virus induced host responses that contribute to disease. In both the cotton rat and pHAECs, the CX4C mutation decreased virus replication and disease and/or host responses to infection. Thus, this mutation, or other mutations that block binding to CX3CR1, has the potential to improve a live attenuated RSV vaccine by attenuating both infection and disease pathogenesis.

Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

Crowd on a Chip: Label-Free Human Monoclonal Antibody Arrays for Serotyping Influenza.

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in "universal" flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Antigenic cartography of H1N1 influenza viruses using sequence-based antigenic distance calculation.

BACKGROUND: The ease at which influenza virus sequence data can be used to estimate antigenic relationships between strains and the existence of databases containing sequence data for hundreds of thousands influenza strains make sequence-based antigenic distance estimates an attractive approach to researchers. Antigenic mismatch between circulating strains and vaccine strains results in significantly decreased vaccine effectiveness. Furthermore, antigenic relatedness between the vaccine strain and the strains an individual was originally primed with can affect the cross-reactivity of the antibody response. Thus, understanding the antigenic relationships between influenza viruses that have circulated is important to both vaccinologists and immunologists. RESULTS: Here we develop a method of mapping antigenic relationships between influenza virus stains using a sequence-based antigenic distance approach (SBM). We used a modified version of the p-all-epitope sequence-based antigenic distance calculation, which determines the antigenic relatedness between strains using influenza hemagglutinin (HA) genetic coding sequence data and provide experimental validation of the p-all-epitope calculation. We calculated the antigenic distance between 4838 H1N1 viruses isolated from infected humans between 1918 and 2016. We demonstrate, for the first time, that sequence-based antigenic distances of H1N1 Influenza viruses can be accurately represented in 2-dimenstional antigenic cartography using classic multidimensional scaling. Additionally, the model correctly predicted decreases in cross-reactive antibody levels with 87% accuracy and was highly reproducible with even when small numbers of sequences were used. CONCLUSION: This work provides a highly accurate and precise bioinformatics tool that can be used to assess immune risk as well as design optimized vaccination strategies. SBM accurately estimated the antigenic relationship between strains using HA sequence data. Antigenic maps of H1N1 virus strains reveal that strains cluster antigenically similar to what has been reported for H3N2 viruses. Furthermore, we demonstrated that genetic variation differs across antigenic sites and discuss the implications.

Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

The induction of antibodies specific for the influenza HA protein stalk domain is being pursued as a universal strategy against influenza virus infections. However, little work has been done looking at natural or induced antigenic variability in this domain and the effects on viral fitness. We analyzed human H1 HA head and stalk domain sequences and found substantial variability in both, although variability was highest in the head region. Furthermore, using human immune sera from pandemic A/California/04/2009 immune subjects and mAbs specific for the stalk domain, viruses were selected in vitro containing mutations in both domains that partially contributed to immune evasion. Recombinant viruses encoding amino acid changes in the HA stalk domain replicated well in vitro, and viruses incorporating two of the stalk mutations retained pathogenicity in vivo. These findings demonstrate that the HA protein stalk domain can undergo limited drift under immune pressure and the viruses can retain fitness and virulence in vivo, findings which are important to consider in the context of vaccination targeting this domain.

Novel Sequence-Based Mapping of Recently Emerging H5NX Influenza Viruses Reveals Pandemic Vaccine Candidates.

Recently, an avian influenza virus, H5NX subclade 2.3.4.4, emerged and spread to North America. This subclade has frequently reassorted, leading to multiple novel viruses capable of human infection. Four cases of human infections, three leading to death, have already occurred. Existing vaccine strains do not protect against these new viruses, raising a need to identify new vaccine candidate strains. We have developed a novel sequence-based mapping (SBM) tool capable of visualizing complex protein sequence data sets using a single intuitive map. We applied SBM on the complete set of avian H5 viruses in order to better understand hemagglutinin protein variance amongst H5 viruses and identify any patterns associated with this variation. The analysis successfully identified the original reassortments that lead to the emergence of this new subclade of H5 viruses, as well as their known unusual ability to re-assort among neuraminidase subtypes. In addition, our analysis revealed distinct clusters of 2.3.4.4 variants that would not be covered by existing strains in the H5 vaccine stockpile. The results suggest that our method may be useful for pandemic candidate vaccine virus selection.

Boolean Modeling of Cellular and Molecular Pathways Involved in Influenza Infection.

Systems virology integrates host-directed approaches with molecular profiling to understand viral pathogenesis. Self-contained statistical approaches that combine expression profiles of genes with the available databases defining the genes involved in the pathways (gene-sets) have allowed characterization of predictive gene-signatures associated with outcome of the influenza virus (IV) infection. However, such enrichment techniques do not take into account interactions among pathways that are responsible for the IV infection pathogenesis. We investigate dendritic cell response to seasonal H1N1 influenza A/New Caledonia/20/1999 (NC) infection and infer the Boolean logic rules underlying the interaction network of ligand induced signaling pathways and transcription factors. The model reveals several novel regulatory modes and provides insights into mechanism of cross talk between NFκB and IRF mediated signaling. Additionally, the logic rule underlying the regulation of IL2 pathway that was predicted by the Boolean model was experimentally validated. Thus, the model developed in this paper integrates pathway analysis tools with the dynamic modeling approaches to reveal the regulation between signaling pathways and transcription factors using genome-wide transcriptional profiles measured upon influenza infection.

Directed selection of influenza virus produces antigenic variants that match circulating human virus isolates and escape from vaccine-mediated immune protection.

Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono-specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non-infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real-world drifted viruses, helping in annual vaccine re-formulation.