Research Project: Intervention Strategies to Control Endemic and New and Emerging Influenza A Virus Infections in Swine

Location: Virus and Prion Research

2022 Annual Report

Objectives
Objective 1. Characterize the ecology, epidemiology, and pathogenesis of emerging swine Influenza A viruses (IAVs) with a focus on the One-Health concept. 1.1. Characterize the pathogenesis, determine the course of infection and evaluate the virulence, focusing on the hemagglutinin, of new and emerging swine IAVs that have the potential to impact swine health and/or affect public health. 1.2. Conduct genetic and antigenic characterization of new and emerging swine IAVs, including phylogenetics and network analysis. 1.3. Identify the molecular mechanisms by which non-swine adapted viruses infect and adapt to swine. 1.4. Using computational methods, characterize new and emerging swine IAVs with regard to entire genetic background (HA, NA and other 7 genes), that have the potential to impact swine health and/or affect public health. 2. Develop intervention strategies to effectively control endemic swine IAVs, including new emerging strains associated with disease outbreaks. 2.1. Enhance virus control and recovery strategies by elucidating the environmental ecology of swine IAVs. 2.2. Characterize the effect of vaccine induced immunity on swine IAV evolution. 2.3. Evaluate and improve existing and new diagnostic tests and testing strategies for swine IAV surveillance, detection, and recovery from disease outbreaks. 2.4. Characterize swine innate and adaptive immune responses to swine IAVs and determine correlates of protection. 2.5. Investigate and develop new vaccine platforms that improve broad cross-protection, override interference from prior immunity, and rapidly control and respond to new and emerging IAV outbreaks in the various components of swine production.

Approach
Influenza A virus (IAV) will be investigated in swine or relevant in vitro models to 1) understand the genetic predictors of host range and virulence in swine; 2) understand the genetic and antigenic variability of endemic viruses and how this affects vaccine strain selection and efficacy; and 3) develop new vaccines that can override maternally-derived antibody interference and provide broader cross-protection. Disease pathogenesis, transmission, and vaccine efficacy studies will be conducted in the natural swine host. Knowledge obtained will be applied to break the cycle of transmission through development of better vaccines or other novel intervention strategies. Computational biology methods will be used to evaluate virus evolution in the natural host to enable predictions to be made on virulence and/or antigenic factors. These predictions will be tested in the lab and in animal studies with wild type viruses and through the use of reverse engineering and mutational studies to identify virulence components of IAV. Experimentally mutated viruses will be evaluated by test parameters that measure both virus and host properties. Development of vaccines that provide better cross-protective immunity than what is currently available with today's vaccines will be approached through understanding correlates of protection, the impact of prior exposure or passive immunity, and through vaccine vector platform development, attenuated strains for vaccines, and other novel vaccine technologies.

Progress Report
In support of Objective 1, Subobjective 1.1, to characterize and evaluate the virulence of endemic and emerging swine IAVs and their impact on public health, genetically representative viruses of circulating swine IAV clades were selected using phylogenetic analyses. The representative viruses were characterized against a panel of monovalent antisera generated against reference swine strains, human seasonal vaccine strains, or candidate vaccine viruses to assess risk of swine-to-human transmission. A swine-to-ferret transmission study was initiated with representative strains to evaluate swine IAV pandemic potential. A pathogenesis and transmission study was performed in pigs to evaluate the risk of strains from the current highly pathogenic avian influenza virus outbreak in the U.S. In support of Objective 1, Subobjective 1.2, to conduct genetic and antigenic characterization of new and emerging swine IAVs, including phylogenetics and network analysis, comprehensive epidemiological analyses were conducted on publicly available swine influenza A virus genes. A one letter code for each internal gene was used to designate the genetic constellation of a strain and paired with hemagglutinin (HA) and neuraminidase (NA) phylogenetic clade combinations. Constellation detection frequencies were analyzed to identify significant changes in spatial and temporal genetic diversity to determine when, where, and for how long IAV phylogenetic clades and reassorted viruses persist. Representative strains were selected to characterize antigenic evolution of contemporary swine HA and NA. In support of Objective 1, Subobjective 1.4, using computational methods, characterize new and emerging swine IAVs with regard to entire genetic background (HA, NA and 6 other genes), that have the potential to impact swine health and/or affect public health, a novel sampling algorithm was developed that objectively selects the most representative IAV strains. The algorithm identifies “spheres” of swine IAV genetic diversity across each IAV genetic phylogeny, and selects strains that minimize genetic distance to all other strains. Representative viruses detected with this algorithm were selected for additional in vitro and in vivo analysis using swine and ferret antisera. A newly emerging H3 clade of viruses that had acquired a novel nucleoprotein (NP) gene segment via reassortment was characterized. Using a reverse-genetics system, an NP gene from the successful H3 clade was inserted into an ancestral H3 strain and a transmission and pathology study in pigs was initiated. A minigenome assay was refined and implemented to assess the replication kinetics and role of genome diversity of IAV in swine with differing whole genome gene constellations. In support of Objective 2, Subobjective 2.3, to evaluate and improve existing and new diagnostic tests and testing strategies for swine IAVs surveillance, detection, and recovery from disease outbreaks, a sampling algorithm was developed to process and visualize the diversity of IAV strains based on user-provided criteria. These automated systems are necessary to account for the increasing volume of IAV in swine sequence data. Automated pipelines (octoFLU) and a graph database (octoFLUdb) were generated to classify the evolutionary lineage and genetic clade of query gene segments. A graphical web interface was deployed and maintained on a public-facing website (https://flu-crew.org) to facilitate rational vaccine design by identifying spatial and temporal trends in genetic diversity in swine IAV sequence data (octoFLUshow). In support of Objective 2, Subobjective 2.4, to characterize swine innate and adaptive immune responses to swine IAVs and determine correlates of protection an animal study was conducted, and swine respiratory tissues were analyzed. Following challenge with IAV, tissues were assessed to measure innate and adaptive immune responses. Immunohistochemical staining techniques were developed for the following host proteins: Mucin 5B (MUC5B; innate), Mucin 5AC (MUC5AC; innate), Mucin 1 (MUC1; innate), Mucin 4 (MUC4; innate), CD163 (macrophage marker; innate), CD3 (lymphocyte marker; adaptive), SLA-II (antigen presentation marker; adaptive), and PAX5 (B cell marker; adaptive). Host gene expression profiles were examined using transcriptomic sequencing techniques from pigs infected with influenza A virus (IAV) from a previously completed study. In support of Objective 2, Subobjective 2.5, to investigate and develop new vaccine platforms that improve broad cross-protection, override interference from prior immunity, and rapidly control and respond to new and emerging IAV outbreaks in the various components of swine production, work continued to evaluate a live attenuated influenza virus vaccine engineered to carry immunomodulatory genes to improve heterologous protection. The role of the neuraminidase in vaccine efficacy against homologous and heterologous challenge strains was evaluated using different vaccine platforms.

Accomplishments
1. Coevolution of influenza surface proteins results in rapid diversification and spatial spread of novel IAV. Vaccine strategies to control IAV infection have focused on the hemagglutinin (HA) protein, but efficacy is challenged by continual genetic change that interferes with vaccine-induced or prior infection immunity. An approach to increase the breadth and depth of vaccine protection is to include a neuraminidase (NA) protein in the vaccine that reflects the diversity of genes circulating in swine. ARS scientists in Ames, Iowa, genetically characterized the N2 subtype NA genes from IAV circulating between 2010 and 2018 in U.S. swine along with their paired HA genes. These data revealed increases in the diversity of the N2 gene, with continual circulation of multiple genetic groups. Interstate movement of pigs and their viruses resulted in rapid changes in genetic diversity of IAV, along with the introduction of novel genes into new geographic regions. This study developed new classifications for N2 genes, demonstrated how NA-HA evolution are paired, and provided critical information for manufacturers and producers on how to objectively develop better vaccines with field-relevant NA components.

2. Detection of new H3N2 influenza A viruses in swine derived from a unique human-to-swine interspecies transmission event. Identifying emerging IAV in swine is important for diagnosis and control of this important respiratory pathogen. In this work, ARS scientists in Ames, Iowa, in collaboration with scientists at the Iowa State University Veterinary Diagnostic Laboratory, identified and characterized a new H3N2 IAV circulating in swine that became established after interpecies transmission of a human seasonal H3N2 from the 2016-17 influenza season. The novel H3.2010.2 viruses transmitted and adapted to the swine host and demonstrated reassortment with internal genes from swine endemic strains, but maintained human-like HA and NA. The novel swine viruses are antigenically distinct from the H3.2010.1 H3N2 transmitted from humans to swine earlier in the 2010 decade. Human-seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection by veterinarians and vaccine manufacturers to improve the ability to prevent respiratory disease in swine as well as the risk of transmission of IAV between species.

3. A method to rapidly analyze and detect novel reassortment in influenza A viruses. The identification of novel influenza A viruses (IAV) that contain genes derived from human-, swine-, or avian-origin IAV is critical for controlling infection in swine and identifying animal viruses with pandemic potential for humans. These novel viruses may be undergoing rapid changes in genetic diversity that reduce the efficacy of vaccine control methods, and may also pose a greater risk to humans by facilitating interspecies infection. ARS scientists in Ames, Iowa, in collaboration with computer scientists at Iowa State University developed a computer program that merged the evolutionary history of individual genes into a network of evolutionary relationships among all 8 gene segments of IAV. The accuracy of the program was validated using whole genome swine IAV data with a known evolutionary history that included transmission of human IAV into swine and subsequent reassortment. The computer program was able to detect known reassortment events, along with additional events between divergent circulating swine IAV strains. The development of this computer program provides computational support for swine IAV surveillance as it is able to objectively rank the novelty of swine IAV strains. These data may aid vaccine development through the objective targeting of novel IAV strains and may help reduce the risk of interspecies transmission by identifying viruses that have pandemic potential due to acquisition of novel gene combinations.

4. Development of octoFLUshow: an interactive tool describing the spatial and temporal trends in the genetic diversity of influenza A viruses in U.S. swine. In the United States, influenza A virus (IAV) in swine is passively monitored through a USDA IAV swine surveillance system. The system was established in 2009, and has since tested over 178,000 samples from more than 55,000 swine diagnostic submissions, resulting in more than 9,000 publicly available virus isolates and genetic sequences. A consistent and continued assessment of the genetic diversity of IAV collected as part of the surveillance system can identify spatial and temporal trends in diversity and novel viruses that require additional characterization. ARS scientists in Ames, Iowa, in collaboration with APHIS colleagues, generated a tool for publicly reporting the USDA IAV surveillance sequencing efforts on single gene and whole virus genome levels. The tool, called octoFLUshow, is an interactive visualization platform. It offers a searchable overview of voluntary relationships among all IAV in swine strains collected in the surveillance system from 2009 to present. This tool provides objective measures of genetic diversity, and allows stakeholders to make informed decisions on vaccine design or use, or in the selection of relevant viruses circulating in U.S. swine herds for further characterization.

5. Implementation of web-based genomic epidemiology tools identified the expansion and spread of a re-emerging H3 influenza A virus in swine. The existence of genetically distinct hemagglutinin (HA) genes of influenza A virus in swine (IAV-S) undermines efforts to control the disease. Swine producers use vaccines to control the virus, and their components are selected by identifying the most common HA gene in a farm or a region. In 2019, ARS scientists in Ames, Iowa, identified an increase in detection frequency of an H3 subtype HA genetic group, C-IVA, in U.S. swine, which was previously circulating at low levels. This study identified genetic and antigenic factors contributing to its resurgence by linking comprehensive evolutionary analyses with antigenic characterization and visualized these analyses in an online genomic epidemiology interface called Nextstrain for IAV in swine. The recently resurging C-IVA viruses did not have a prior increase in genetic diversity nor significant HA or NA antigenic changes. Instead, many of the contemporary C-IVA IAV viruses acquired a novel internal gene segment via reassortment with human seasonal H1N1 that might have contributed to the genetic group's success. These data demonstrated how surveillance can detect when minor populations of genetically diverse IAV in swine persist, and subsequently sweep across the landscape by infecting populations of animals that do not have vaccine-induced or prior infection immunity, enabling veterinarians and vaccine manufacturers to develop targeted vaccines to control IAV transmission.

6. The global genetic and antigenic diversity of influenza A virus in swine detected between January and December 2021. H1N1, H1N2, and H3N2 influenza A virus (IAV) subtypes are endemic in swine herds around the world and characterizing the genetic and antigenic diversity of these viruses can provide rational criteria for control efforts and informing public health initiatives. Because of the risk animal IAV pose to the human population, experts at the World Health Organization (WHO) vaccine composition meeting review cases of humans infected with animal IAV and consider them for development of pandemic-preparedness candidate vaccine viruses (CVV). ARS scientists in Ames, Iowa, in collaboration with the joint World Organization for Animal Health (WOAH) and Food and Agriculture Organization of the United Nations (FAO) scientific network on animal influenza, OFFLU, quantified the global genetic diversity of swine IAV circulating across two reports spanning January to December 2021. The circulating swine IAV was compared to human IAV vaccines and current candidate vaccine viruses (CVV) that are used for pandemic preparedness, and representative swine IAV were antigenically characterized using a panel of anti-sera against human vaccine strains or CVV strains. The data demonstrated 19 genetically distinct cocirculating swine IAV groups. Twenty-one human cases with IAV of swine origin were identified and linked to nine of the 19 swine genetic groups. Fifteen of the 19 distinct swine genetic groups had reduced antibody recognition by CVV or vaccine strain antisera, identifying gaps of coverage by human pandemic preparedness vaccines. Seven of the 15 groups have a history of known transmission from swine to humans. These analyses demonstrate the dynamic interplay of IAV transmission between humans and swine and identified genetic groups that are considered by the WHO to improve pandemic preparedness efforts.

7. Vaccine-associated enhanced respiratory disease following influenza A virus infection in ferrets recapitulated the model previously identified in pigs. Results described vaccine associated enhanced respiratory disease (VAERD) and this model provides an additional tool to study influenza vaccine safety and efficacy. Prior to this report, research described VAERD in swine, but it was not well-characterized in other influenza host species such as ferrets. Influenza A viruses in swine are highly diverse, and although vaccines are the best method to prevent influenza illness in swine, mismatched vaccines of the whole inactivated virus platform are associated with VAERD. ARS scientists in Ames, Iowa, demonstrated the susceptibility of ferrets, a common model species of human influenza infection, to VAERD using an experimental model of VAERD previously demonstrated in pigs and showed that the clinical disease between the two host species were similar. The induction of VAERD in ferrets highlights the potential risk in humans and the need to consider VAERD when designing and evaluating vaccine strategies as an additional tool to study influenza vaccine safety and efficacy for vaccine manufacturers and researchers.

8. Antigenic distance between North American H3N2 influenza A viruses in swine and human seasonal H3N2 influenza A viruses as an indication of zoonotic risk to humans. Human H3N2 influenza A viruses (IAV) spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation of H3N2 in swine and increased IAV diversity in pig populations. The evolution in swine H3N2 led to a reduced similarity with human seasonal H3N2 and the vaccine strains used to protect human populations. ARS scientists in Ames, Iowa, found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the previous decade of incursion but had substantial difference compared with more recent human vaccine strains. Additionally, pandemic preparedness vaccine strains developed for public health were also less similar to contemporary swine strains. Lastly, post-exposure and post-vaccination human sera revealed that although antibodies were detected against human H3N2 strains, many had limited immunity to swine H3N2, particularly the swine viruses derived from 1990s transmission events, especially in older adults born before 1970. This is likely due to a skewed immune response to those human seasonal H3N2 that circulated between 1970 and 1990. These antigenic assessments of swine H3N2 provide critical information for pandemic preparedness and candidate vaccine development.

9. Evolution and antigenic advancement of N2 neuraminidase of swine influenza a viruses circulating in the United States following two separate introductions from human seasonal viruses. Vaccine strategies to control influenza A virus (IAV) infection focus on the hemagglutinin (HA) protein, but efficacy is challenged by continual genetic change that interferes with vaccine-induced immunity or prior infection immunity. An approach to increase the breadth and depth of vaccine protection is to include a neuraminidase (NA) protein that reflects the diversity of genes circulating in swine. The NA gene of the N2 subtype currently accounts for approximately two-thirds of the NA detections in US domestic swine populations. The N2 genes have undergone substantial genetic and antigenic evolution following introductions of human seasonal H3N2 subtype into swine in 1998 and human seasonal H1N2 subtype in 2002. Increased genetic diversity of the N2 genes suggested an increase in antigenic evolution of the virus surface glycoprotein coded by the genes, and these changes may allow escape from natural and/or vaccine induced immunity. To assess the potential loss in immune recognition among naturally occurring N2 proteins from swine, ARS scientists in Ames, Iowa, characterized the genetic evolutionary distance and the antigenic distance between wild-type swine N2 IAV viruses and then selecting and generating a panel of antisera against representative N2. In the 20+ years following the introduction, the genetic diversity of N2 genes in swine increased. This corresponded with an increase in antigenic diversity. Antibodies generated against representative N2 of the 1998 N2 did not react with N2 protein of the 2002 lineage and vice versa. Further, both the 1998 and 2002 N2 lineages displayed antigenic changes over time, indicating the N2 in IAV of U.S. swine harbors a substantial amount of antigenic diversity.Understanding NA genetic and antigenic diversity in swine IAV has important implications for effective vaccine design and including an N2 component in vaccines that matches circulating diversity will likely improve vaccine efficacy and reduce the impact of IAV.

Review Publications
Arendsee, Z.W., Chang, J., Hufnagel, D.E., Markin, A., Baker, A.L., Anderson, T.K. 2021. octoFLUshow: an interactive tool describing spatial and temporal trends in the genetic diversity of influenza A virus in U.S. swine. Microbiology Resource Announcements. 10(50). Article e01081-21. https://doi.org/10.1128/MRA.01081-21.
Neveau, M.M., Zeller, M.A., Kaplan, B.S., Souza, C.K., Gauger, P.C., Baker, A.L., Anderson, T.K. 2022. Genetic and antigenic characterization of an expanding H3 influenza A virus clade in US swine visualized by Nextstrain. mSphere. 7(3). Article 00994-21. https://doi.org/10.1128/msphere.00994-21.
Zeller, M.A., Chang, J., Baker, A.L., Gauger, P.C., Anderson, T.K. 2021. Spatial and temporal coevolution of N2 neuraminidase and H1 and H3 hemagglutinin genes of influenza A virus in US swine. Virus Evolution. 7(2). Article veab090. https://doi.org/10.1093/ve/veab090.
Sharma, A., Zeller, M.A., Souza, C.K., Anderson, T.K., Baker, A.L., Harmon, K., Li, G., Zhang, J., Gauger, P.C. 2022. Characterization of a 2016-2017 human-seasonal H3 influenza A virus spillover now endemic to U.S. swine. mSphere. 7(1). Article e00809-21. https://doi.org/10.1128/msphere.00809-21.
Kaplan, B.S., Anderson, T.K., Chang, J., Santos, J., Perez, D., Lewis, N., Vincent, A.L. 2021. Evolution and antigenic advancement of N2 neuraminidase of swine influenza A viruses circulating in the United States following two separate introductions from human seasonal viruses. Journal of Virology. 95(20). https://journals.asm.org/doi/10.1128/JVI.00632-21.
Souza, C.K., Anderson, T.K., Chang, J., Venkatesh, D., Lewis, N.S., Pekosz, A., Shaw-Saliba, K., Rothman, R.E., Chen, K., Baker, A.L. 2022. Antigenic distance between North American swine and human seasonal H3N2 influenza A viruses as an indication of zoonotic risk to humans. Journal of Virology. 96(2). Article e01374-21. https://doi.org/10.1128/JVI.01374-21.
Anderson, T.K., Inderski, B., Diel, D.G., Hause, B.M., Porter, E., Clement, T., Nelson, E.A., Bai, J., Lager, K.M., Faaberg, K.S., Christopher-Hennings, J., Gauger, P.C., Zhang, J., Harmon, K.M., Main, R. 2021. The United States Swine Pathogen Database: Integrating veterinary diagnostic laboratory sequence data to monitor emerging pathogens of swine. Database: The Journal of Biological Databases and Curation. 2021. Article baab078. https://doi.org/10.1093/database/baab078.
Nicholson, T.L., Waack, U., Anderson, T.K., Bayles, D.O., Zaia, S.R., Goertz, I., Eppinger, M., Hau, S.J., Brockmeier, S., Shore, S. 2021. Comparative virulence and genomic analysis of streptococcus suis isolates. Frontiers in Microbiology. 11. Article 620843. https://doi.org/10.3389/fmicb.2020.620843.
Markin, A., Wagle, S., Anderson, T.K., Eulenstein, O. 2022. RF-Net 2: Fast inference of virus reassortment and hybridization networks. Bioinformatics. 38(8):2144-2152. Article btac075. https://doi.org/10.1093/bioinformatics/btac075.
Staton, M., Cannon, E.K., Sanderson, L., Wegrzyn, J., Buehler, S., Ficklin, S., Grau, E., Guignon, V., Gunoskey, J., Jung, S., Main, D., Poelchau, M.F., Ramnath, R., Cobo, I., Richter, P., West, J., Anderson, T.K., Inderski, B., Faaberg, K.S., Lager, K.M. 2021. Tripal, a community update after 10 years of supporting open source, standards-based genetic, genomic and breeding databases. Briefings in Bioinformatics. 22(6). https://doi.org/10.1093/bib/bbab238.
Sitthicharoenchai, P., Burrough, E., Arruda, B.L., Harmon, K., Bradner, L., Magstadt, D., Burrough, E., Derscheid, R., Michael, A., Nunez De Almeida, M., Schumacher, L., Siepker, C., Stevenson, G. 2021. Comparative analysis of novel strains of porcine astrovirus type 3 in the USA. Viruses. 13(9). Article 1859. https://doi.org/10.3390/v13091859.
Kimble, B.J., Brand, M.W., Kaplan, B.S., Coyle, E.M., Chilcote, K., Gauger, P., Khurana, S., Baker, A.L. 2022. Vaccine-associated enhanced respiratory disease following influenza virus infection in ferrets recapitulates the model in pigs. Journal of Virology. 96(5). https://doi.org/10.1128/jvi.01725-21.