Influenza Research Database: An integrated bioinformatics resource for influenza virus research

The Influenza Research Database (IRD) is a U.S. National Institute of Allergy and Infectious Diseases (NIAID)-sponsored Bioinformatics Resource Center dedicated to providing bioinformatics support for influenza virus research. IRD facilitates the research and development of vaccines, diagnostics and therapeutics against influenza virus by providing a comprehensive collection of influenza-related data integrated from various sources, a growing suite of analysis and visualization tools for data mining and hypothesis generation, personal workbench spaces for data storage and sharing, and active user community support. Here, we describe the recent improvements in IRD including the use of cloud and high performance computing resources, analysis and visualization of user-provided sequence data with associated metadata, predictions of novel variant proteins, annotations of phenotype-associated sequence markers and their predicted phenotypic effects, hemagglutinin (HA) clade classifications, an automated tool for HA subtype numbering conversion, linkouts to disease event data and the addition of host factor and antiviral drug components. All data and tools are freely available without restriction from the IRD website at https://www.fludb.org.National Institutes of Health/National Institute for Allergy and Infectious Diseases [HHSN272201400028C]. Funding for open access charge: J. Craig Venter Institute

Genetic and Antigenic Evolution of Influenza A (H3N2) Virus Neuraminidase

There is still much uncertainty about the underlying mechanisms that govern antigenic drift of influenza viruses, and several theories have been proposed. These theories consider hemagglutinin (HA) to be the primary driving force, while the second key surface glycoprotein (neuraminidase, NA) and the other viral proteins have largely been ignored. This thesis focuses on all influenza A (H3N2) virus proteins, with special emphasis on NA, to better understand—and ultimately predict—the complex evolution of A(H3N2) viruses. The genetic evolution, with respect to antigenic change, was analyzed by comparing HA and NA (chapter 2) and by studying the whole-genome (chapter 3). We next optimized the enzyme-linked lectin assay (ELLA) as an NA inhibition (NI) assay for human serology (chapter 5) and rapid antigenic characterization (chapter 6). With the optimized NI ELLA, we analyzed NA from 1968 till the 2010-2011 season and subsequently compared it to HA (chapter 7). Our work clearly shows that there is antigenic evolution for NA, thus warranting the inclusion of NAs representing emerging influenza A strains in vaccines. Increasing knowledge on which mutations cause changes in the phenotype of NA can help to perform more targeted influenza surveillance. It would then be advisable to integrate genetic and antigenic NA data with sequence and antigenic data of HA, epidemiological data, and geographical data during influenza surveillance. This will facilitate consideration of NA content, and improve next generation influenza vaccines

CHARACTERIZING THE ROLE OF N TERMINUS OF INFLUENZA A NUCLEOPROTEIN FOR LOCATION AND VIRAL RNP ACTIVITY

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay

Generation and Comprehensive Analysis of Host Cell Interactome of the PA Protein of the Highly Pathogenic H5N1 Avian Influenza Virus in Mammalian Cells

Accumulating data have identified the important roles of PA protein in replication and pathogenicity of influenza A virus (IAV). Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis. In this study, using immunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry, we identified 278 human cellular proteins that might interact with PA of H5N1 IAV. Gene Ontology annotation revealed that the identified proteins are highly associated with viral translation and replication. Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation, infectious disease, and signal transduction. In addition, Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival. Moreover, two cellular proteins (nucleolin and eukaryotic translation elongation factor 1-alpha 1) identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays. Therefore, this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells

Survey and Molecular Typing of Influenza A Virus among Palestinians

Influenza A virus (IAV) causes significant mortality, morbidity, and financial burden throughout the world. IAV is a negative, single-stranded, and segmented RNA virus of the Orthomyxoviridae family. IAV subtypes are determined based on its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). H1N1 and H3N2 are the major circulating subtypes among humans. Frequent genotyping of IAV strains throughout the influenza season is crucial for the identification of circulating subtypes and subsequently the choice of the H3N2 and H1N1 subtypes’ lineages to be included in the annually prepared vaccine cocktail. Sequencing and identification of circulating subtypes in the region continues to be less frequent and less intensive than in other parts of the world, despite increasing interest and efforts made ever since the swine H1N1 outbreak in 2009. This work presents the first comprehensive study on IAV circulating in Palestine. 200 Nasopharyngeal aspirate (NPA) samples were collected between February 10th and May 5th, 2015 from participants suffering from mild to severe symptoms of upper respiratory infections and were screened for the presence of IAV using RT-PCR assays amplifying the HA and NA gene regions. 50 samples (25%) tested positive for IAV, 24 (48%) were identified as H1N1, and 26 (52%) were identified as H3N2 subtype, respectively. Infection with H1N1 occurred mainly in April, while H3N2 infections were mainly detected in March. Most IAV infections in children younger than 6-year-old were attributed to subtypes H3N2, while H1N1 was responsible for most infections in adults older than eighteen-year-old. Hundred-fifteen sequences of the HA and NA genes were successfully analyzed. These sequences belong to 23 IAV positive Palestinian IAV samples. The percent identity of thesequences among Palestinian isolates was higher than that between Palestinian isolates and GenBank-archived reference sequences. 14, 15, 22 and 6 non-synonymous substitutions were detected in the Palestinian H1, N1, H3 and N2 genes, including novel ones, respectively. Some of these amino acid substitutions localized to the antigenic sites, T202S and Q180K in H1 gene, T144A and L173S in H3 gene. Such substitutions in the antigenic sites may affect the hostimmune response. Other substitutions located at the receptor binding sites, such as T228A in H3 gene, may affect viral binding activity to host cell receptor, and subsequently its virulence and host species barrier.None of the substitutions detected in this work were associated with drug resistance or fatal outcomes. Phylogenetic analysis revealed that Palestinian H1N1 and H3N2 were not closely related to those regionally circulating strains and clustered closer to international, rather nonregional strains. The results of this study are significant in providing the first insight into the genetic properties of the HA and NA genes of the influenza A viruses circulating in Palestine. Finally, this study provides evidence of the efficacy of the seasonal influenza vaccine 2014-2015 in Palestine

Purification and proteomics of influenza virions

This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry

Identification of host factors in swine respiratory epithelial cells that contribute to host anti-viral defense and influenza virus replication

Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function