Influenza Virus Research Research Articles

Misclassified: identification of zoonotic transition biomarker candidates for influenza A viruses using deep neural network.

Introduction: Zoonotic transition of Influenza A viruses is the cause of epidemics with high rates of morbidity and mortality. Predicting which viral strains are likely to transition from their genetic sequence could help in the prevention and response against these zoonotic strains. We hypothesized that features predictive of viral hosts could be leveraged to identify biomarkers of zoonotic viral transition. Methods: We trained deep learning models to predict viral hosts based on the virus mRNA or protein sequences. Our multi-host dataset contained 848,630 unique nucleotide sequences obtained from the NCBI Influenza Virus and Influenza Research Databases. Each sequence, representing one gene from one viral strain, was classified into one of the three host categories: Avian, Human, and Swine. Trained models were analyzed using various neural network interpretation methods to identify interesting candidates for zoonotic transition biomarkers. Results: Using mRNA sequences as input led to higher prediction accuracies than amino acids, suggesting that the codon sequence contains information relevant to viral hosts that is lost during protein translation. UMAP visualization of the latent space of our classifiers showed that viral sequences clustered according to their host of origin. Interestingly, sequences from pandemic zoonotic viral strains localized at the margins between hosts, while zoonotic sequences incapable of Human-to-Human transmission localized with non-zoonotic viruses from the same host. In addition, host prediction for pandemic zoonotic sequences had low prediction accuracy, which was not the case for the other zoonotic strains. This supports our hypothesis that ambiguously predicted viral sequences bear features associated with cross-species infectivity. Finally, we compared misclassified sequences to well-classified ones to extract interesting candidates for zoonotic transition biomarkers. While features varied significantly between pairs of species and viral genes, several codons were conserved in Swine-to-Human and Avian-to-Human misclassified sequences, and in particular in the NA, HA, and NP genes, suggesting their importance for zoonosis in Humans. Discussion: Analysis of viral sequences using neural network interpretation approaches revealed important genetic differences between zoonotic viruses with pandemic potential, compared to non-zoonotic viral strains or zoonotic viruses incapable of Human-to-Human transmission.

Hugh John Forster Cairns. 21 November 1922—12 November 2018

John Cairns was a magnet for scientists (and non-scientists) of all types, from undergraduates to Nobel laureates. For him science was fun, and discussing science with others even more fun. Soon after finishing medical school (Oxford) at the end of the Second World War he gravitated towards research, his first publication linking the incidence of penicillin-resistant bacteria with long hospital stays. He then studied virology with Macfarlane Burnet FRS in Melbourne, Australia, after which he repaid his two years of National Service from the Second World War at the Virus Research Institute in Entebbe, Uganda. He returned to Australia (Canberra), continuing influenza virus research. However, while visiting Caltech in the 1950s, he was exposed to the emerging field of molecular biology, of which he became a pioneer. After returning to Canberra from a sabbatical with Alfred Hershey at Cold Spring Harbor in 1961, he showed that the Escherichia coli genome is circular, and demonstrated the replication fork. In 1963 he was appointed director of the Cold Spring Harbor Laboratory (CSHL). He successfully saved CSHL from bankruptcy before handing over directorship to Jim Watson (ForMemRS 1981). At CSHL he proved that the so-called Kornberg DNA polymerase was not required for replication of the E. coli genome. In the 1970s Cairns turned his attention to understanding cancer and moved to North London to direct a small research institute in Mill Hill, a satellite of the Imperial Cancer Research Fund based in central London. Cairns wrote two influential reviews on cancer plus the book Cancer: science and society , seamlessly weaving together information across disciplines, from human population levels to molecular levels, plus everything in between. He fostered multi-disciplinary basic research at the Mill Hill labs, recruiting scientists who used model organisms such as bacteria, fruit flies, slime moulds, amphibians and mice as models to explore the determinants of cell fate during development; such pathways seemed likely to be involved in the transformation of normal cells into cancer cells. Cairns created and fostered an exceptionally stimulating, interactive, nurturing and cutting-edge research environment that launched the careers of many scientists. In his own lab at Mill Hill, Cairns discovered new pathways for DNA alkylation repair in E. coli , and subsequently as professor of cancer biology at the Harvard School of Public Health he discovered what is now known as stress-induced mutagenesis, also in E. coli .

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

The Intersection of Age and Influenza Severity: Utility of Ferrets for Dissecting the Age-Dependent Immune Responses and Relevance to Age-Specific Vaccine Development.

Many factors impact the host response to influenza virus infection and vaccination. Ferrets have been an indispensable reagent for influenza virus research for almost one hundred years. One of the most significant and well-known factors affecting human disease after infection is host age. Another significant factor is the virus, as strain-specific disease severity is well known. Studying age-related impacts on viral infection and vaccination outcomes requires an animal model that reflects both the physiological and immunological changes that occur with human aging, and sensitivity to differentially virulent influenza viruses. The ferret is uniquely susceptible to a plethora of influenza viruses impacting humans and has proven extremely useful in studying the clinical and immunological pictures of influenza virus infection. Moreover, ferrets developmentally have several of the age-related physiological changes that occur in humans throughout infancy, adulthood, old age, and pregnancy. In this review, we discuss ferret susceptibility to influenza viruses, summarize previous influenza studies using ferrets as models of age, and finally, highlight the application of ferret age models in the pursuit of prophylactic and therapeutic agents to address age-related influenza disease severity.

Surface acoustic wave (SAW) real-time interaction analysis of influenza A virus hemagglutinins with sialylated neoglycolipids.

Real-time interaction analysis of H1 hemagglutinin from influenza A H1N1 (A/New York/18/2009) and H7 hemagglutinin from influenza A H7N7 (A/Netherlands/219/03) with sialylated neoglycolipids (neoGLs) was performed using the surface acoustic wave (SAW) technology. The produced neoGLs carried phosphatidylethanolamine (PE) as lipid anchor and terminally sialylated lactose (Lc2, Galβ1-4Glc) or neolactotetraose (nLc4, Galβ1-4GlcNAcβ1-3Galβ1-4Glc) harboring an N-acetylneuraminic acid (Neu5Ac). Using α2-6-sialylated neoGLs, H1 and H7 exhibited marginal attachment toward II6Neu5Ac-Lc2-PE, whereas Sambucus nigra lectin (SNL) exhibited strong binding and Maackia amurensis lectin (MAL) was negative in accordance with their known binding preference toward a distal Neu5Acα2-6Gal- and Neu5Acα2-3Gal-residue, respectively. H1 revealed significant binding toward IV6Neu5Ac-nLc4-PE when compared to weak interaction of H7, whereas SNL showed strong and MAL no attachment corresponding to their interaction specificities. Additional controls of MAL and SNL with α2-3-sialylated II3Neu5Ac-Lc2-PE and IV3Neu5Ac-nLc4-PE underscored the reliability of the SAW technology. Pre-exposure of model membranes spiked with α2-6-sialylated neoGLs to Vibrio cholerae neuraminidase substantially reduced the binding of the hemagglutinins and the SNL reference. Collectively, the SAW technology is capable of accurate measuring binding features of hemagglutinins toward neoGL-spiked lipid bilayers, which can be easily loaded to the functionalized biosensor gold surface thereby simulating biological membranes and suggesting promising clinical application for influenza virus research.

Universal anti-influenza vaccines based on viral HA2 and M2e antigens.

Aquatic birds are the main reservoir of influenza A viruses (IAVs). These viruses can infect humans repeatedly and cause acute respiratory disease with potential of spread in the form of epidemics. In addition, avian influenza viruses that overcome the interspecies barrier and adapt to humans can cause a world-wide pandemic with severe consequences to human health. Therefore, scientists are focused on the development of a "universal" vaccine with a broad protective efficacy, i.e. against different subtypes of influenza A viruses and not only against the currently co-circulating human epidemic strains. Nowadays, several new vaccine design strategies have been described. Most of them utilize the conserved stem part of influenza surface glycoprotein hemagglutinin (HA) or the ectodomain of M2 (M2e) protein with proton-channel activity. A comparison of the efficacy of novel vaccines and their protective mechanisms against influenza infection is discussed in this review and should be considered for the construction of the most effective broadly protective vaccine with minimal side effects. This is the essential goal in influenza virus research today, especially when the infection with new human coronavirus SARS-CoV-2 can interfere with the course of influenza virus infection. Keywords: influenza A virus; HA2 gp; M2 ectodomain; universal vaccine.

Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses.

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

Avian H5N1 influenza virus infection causes severe pneumonia in the Northern tree shrew (Tupaia belangeri)

Avian-origin influenza viruses like H5N1 and H7N9 often cause severe symptoms with high mortality in humans. Animal models are useful for clarification of the mechanisms of pathogenicity of these infections. In this study, to expand the potential utility of the Northern tree shrew (Tupaia belangeri) for influenza virus infection, we assessed the pathogenicity of H5N1 and H7N9 avian influenza viruses in tupaia. Infectious virus was detected continuously from nasal, oral, tracheal, and conjunctival swab samples in the animals infected with these viruses. H5N1 influenza virus infection of tupaia caused severe diffuse pneumonia with fever and weight loss. In contrast, H7N9 influenza virus infection caused focal pneumonia. The severity of pneumonia was correlated with proinflammatory cytokine transcript levels. These results indicated that tupaia can be another suitable animal model for avian influenza virus research.

Moving Forward: Recent Developments for the Ferret Biomedical Research Model.

Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. However, there are limitations to the model that must be overcome for maximal utility for the scientific community. Here, we describe important recent advances that will accelerate biomedical research with this animal model.

Assessment of Influenza Virus Hemagglutinin Stalk-Specific Antibody Responses.

Animal models are essential to examine the pathogenesis and transmission of influenza viruses and for preclinical evaluation of influenza virus vaccines. Among the animal models used in influenza virus research, the domestic ferret (Mustela putorius furo) is the gold standard. As seen in humans, infection with influenza virus or immunization with an influenza virus vaccine induces humoral and cellular immunity in ferrets that provides protection against infection by an antigenically similar influenza virus. Antibodies against the globular head domain of the influenza hemagglutinin can provide sterilizing immunity against virus infection by blocking receptor binding. However, antibodies that bind the stalk region of the hemagglutinin also confer protection by several mechanisms including antibody-dependent cellular cytotoxicity or phagocytosis. Recently, the antigenically and structurally conserved hemagglutinin stalk has become an attractive target for the development of universal influenza virus vaccines that hold the promise to provide protection against influenza epidemics and pandemics. Herein, in vivo and in vitro assays, including optimization of assay conditions to examine hemagglutinin stalk-specific antibody responses in small animal models, are described.

Measuring Influenza Virus Infection Using Bioluminescent Reporter Viruses for In Vivo Imaging and In Vitro Replication Assays.

To streamline standard virological assays, we developed bioluminescent replication-competent influenza reporter viruses that mimic their parental counterparts. These reporter viruses provide a rapid and quantitative readout of viral infection and replication. Moreover, they permit real-time in vivo measures of viral load, tissue distribution, and transmission in the same cohort of animals over the entire course of infection-measurements that were not previously possible. Here we provide detailed protocols using bioluminescent reporter viruses for in vivo imaging in mice and ferrets. We also describe cell culture-based techniques using reporter viruses for quantification of viral titers and performing microneutralization assays. The ease, speed, and adaptability of these approaches have the potential to accelerate multiple areas of influenza virus research.

Generation of a serum free CHO DG44 cell line stably producing a broadly protective anti-influenza virus monoclonal antibody.

Because of the broad neutralization and in vivo protection across influenza A and influenza B virus strains, monoclonal antibody CR9114 is widely used in influenza virus research as a positive control in many experiments. To produce amounts sufficient for the demand requires regular transient transfections, resulting in varying yield as well as differing batch to batch quality. Here, we report the development of a serum-free CHO DG44 cell line, stably producing a CR9114-like antibody with a potential to become a useful influenza virus research tool.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication.

ABSTRACTInfluenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication.

Distinct susceptibility and applicability of MDCK derivatives for influenza virus research.

Madin-Darby Canine Kidney (MDCK) cells are widely utilized as a substrate for influenza virus isolation and propagation due to the high yields of virus. Here we compared the conventional MDCK cell line, MDCK-SIAT1 and MDCK-London for viral production, cell survival, and suitability in testing antivirals using six influenza strains including two H1N1 (pandemic and epidemic strains), three H3N2 and one influenza B strain. Overall our results suggest that MDCK-London cell line is superior for virus culturing and quantification, and hence an ideal platform to evaluate antiviral drug efficacy against multiple strains of influenza. Our data also suggests that while virus titers determined by the hemagglutination assay (HA) and neuraminidase activity (NA) are widely used to indicate viral load, there is a poor correlation between these measurements and the infectious titer obtained by plaque assay.

Influenza Virus Research and Eu Export Regulations: Publication, Proliferation, and Pandemic Risks.

An influenza pandemic would be a global health emergency, and laboratory-based research on influenza viruses is an important component of worldwide efforts to prevent and prepare for this. There are concerns, however, that publishing the findings of such research might sometimes increase the risk of a pandemic caused by a laboratory accident or the deliberate release of a deadly virus. This article addresses the challenge of governing scientific information sharing, with regard to public health benefits and risks, from an export-control perspective. The discussion focuses on research findings produced in 2011 by a team of influenza virologists in the Netherlands, and on the Dutch Government’s unprecedented decision to regard the intended publication of these findings as being subject to European Union regulations on the export of ‘dual-use’ items. I argue that, when a government is uncertain about whether the benefits of publishing particular research findings in a scientific journal outweigh the risks, a process of selectively disseminating those findings should be available as an alternative to official censorship.

Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

Avian influenza viruses pose a serious zoonotic threat, in part because current seasonal influenza virus vaccines only offer strain-specific protection, instead of heterosubtypic or universal protection against influenza virus infection. Understanding the humoral response to vaccination and natural infection in the broadest context possible is important to developing defenses against influenza zoonosis. Protein microarrays are a novel platform well suited to assaying the humoral immune response broadly and efficiently. We developed an influenza virus protein microarray (IVPM) that could be used to assay sera from many species, including humans. Waterfowl such as mallard ducks are natural reservoirs for many influenza A viruses, but their humoral immune response to infection is poorly understood. To establish this technology, we assayed sera from mallards experimentally infected with two low-pathogenic common avian influenza viruses (H3N8 and H4N5) for reactivity to influenza virus hemagglutinin (HA) by IVPM. The IVPM results correlated well with results from an established enzyme-linked immunosorbent assay, supporting the validity of the IVPM as a serological assay in influenza virus research. Interestingly, successive infection with H3N8 followed by H4N5 virus in mallard ducks induced antibodies that were broadly reactive against group 2 hemagglutinins. We also analyzed sera from wild mallards and observed serological evidence for infection in those sera. With serological information, it may be possible to infer infection history of wild avian species and gain a better understanding of the infection dynamics of influenza viruses in their natural reservoir. This might ultimately lead to interventions that enhance our pandemic preparedness.Emerging Microbes & Infections (2017) 6, e110; doi:10.1038/emi.2017.98; published online 6 December 2017

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.

Filamentous influenza viruses

Clinical isolates of influenza virus produce pleomorphic virus particles, including extremely long filamentous virions. In contrast, strains of influenza that have adapted to laboratory growth typically produce only spherical virions. As a result, the filamentous phenotype has been overlooked in most influenza virus research. Recent advances in imaging and improved animal models have highlighted the distinct structure and functional relevance of filamentous virions. In this review we summarize what is currently known about these strikingly elongated virus particles and discuss their possible roles in clinical infections.

Deep sequencing reveals the viral adaptation process of environment-derived H10N8 in mice

The H10N8 virus was isolated from the water of Dongting Lake, China. Mice were infected while showing no obvious symptoms and replication was restricted to the lungs. When the wild-type virus was serially passaged in the lungs of mice, the resulting viruses became lethal and capable of replication in many other organs. This offered an applicable model for the exploration of viral genome gradual mutation during adaptation in mice. The different passage viruses from mice lung lavage were named P1, P3, P5, and P7, respectively. We sequenced the four viruses using next-generation sequencing (NGS) to analyze the dynamics of the H10N8 viral genome, polymorphism, and amino acid mutation of related proteins. We aimed to demonstrate how a mutant strain of low pathogenicity could become lethal to mice. Using Illumina high-throughput data, we detected the gradual mutations of F277S, C278Q, F611S and L653P in the polymerase acidic (PA) protein, and of L207V and E627K in the PB2 protein during adaptation. Interestingly, many amino acid sites mutated quickly; the others did so more slowly and remained in a heterozygous state for several generations. The PA amino acids S277 and Q278 have previously been found in clinical wild-type strains, including the human-H10N8 isolate in 2013. This demonstrates that the wild-type H10N8 virus had mutated to adapt to mammalian hosts. These data provide important reference information for influenza virus research.