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

Goals / 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.

Project Methods
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 10/01/23 to 09/30/24

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. In support of Objective 1, Subobjective 1.1, to conduct in vitro experiments to identify viruses with risk to swine or between swine and other susceptible species (humans), genetically representative swine viruses were selected using phylogenetic analyses. These panels of swine IAV were characterized for antigenic phenotype, pH of inactivation, and receptor binding profiles using glycan arrays. These are phenotypes that can indicate propensity for human infection. In support of Objective 1, Subobjective 1.2, to integrate genotype and phenotype to generate long-term forecasts of genetic clade persistence, turnover, and emergence. A novel algorithm was developed that determines how different clusters of genes are linked. The method allows different influenza A virus gene trees to be merged to generate a single hypothesis on the evolutionary history of the virus while accounting for reassortment. An evolutionary study on H1N1, H1N2, and H3N2 whole genome sequence data was performed to determine the preferential pairings between different gene segments based on the frequency of reassortment among the segments. In support of Objective 1, Subobjective 1.3, to identify HA or NA or polymerase factors for adaptation in swine vs. non-swine adapted IAV in vitro, minigenome assays to assess the role of different polymerase genes from swine IAV in replication efficiency were conducted. Because of an ongoing H5N1 highly pathogenic avian influenza (HPAI) outbreak, additional pathogenesis and transmission studies were performed in pigs with clade 2.3.4.4b H5N1 HPAI viruses. Multiple strains with different genotypes and with or without mammalian adaptation markers were evaluated. Human seasonal H3N2 were detected in swine and a representative isolate evaluated in a pathogenesis and transmission study in pigs. In support of Objective 2, Subobjective 2.1, to evaluate the role of seasonality, timing, and climate drivers of influenza A virus in swine, a study was conducted to describe the spatial and temporal diversity of HA and NA genes and reassortment of whole genomes from 2009 to 2022. Regions that acted as sources or sinks for IAV genomic diversity were quantified. The probability of novel IAV genotypes being disseminated was statistically modeled. Each US region harbored a unique diversity of HA, NA, and genome constellations, with region specific patterns. Objective 2, Subobjective 2.4, to identify host genes associated with virulence or phenotypes of IAV, pigs lacking the host protease TMPRSS2 were assessed for susceptibility to a swine 1A.3.3.2 H1N1 virus. In a separate study, pigs lacking the host polymerase co-factor ANP32A were assessed for susceptibility to a clade 2.3.4.4b H5N1 HPAI virus. Artificial Intelligence (AI)/Machine Learning (ML) A non-neural generalized, light-weight machine learning software was developed. The software can be deployed on local computers or HPC clusters for the classification of genetic sequences from surveillance https://github.com/flu-crew/classLog. This approach facilitates the analysis of large volumes of genetic sequence data derived from the national USDA influenza A virus in swine surveillance system. The generalized ML software was integrated into a regional veterinary diagnostic laboratory testing platform and acts as an early-warning system that detects when novel influenza A viruses are isolated in clinical diagnostic submissions. ACCOMPLISHMENTS 01 The exchange of gene segments between influenza A viruses (IAV) is linked to increased transmission efficiency. IAV are composed of eight gene segments that can be exchanged during coinfection of an animal host to create new combinations. Some gene combinations may have a transmission advantage and be paired together preferentially. ARS scientists in Ames, Iowa, in collaboration with investigators at the Iowa State University Veterinary Diagnostic Laboratory detected an exchange of IAV genes in swine populations in the United States that involved two lineages of nucleoprotein (NP) genes. One lineage of the NP gene then became a predominant lineage detected in genomic surveillance. Using an animal transmission study, the exchange of NP genes between viruses was demonstrated to cause the virus to shed from the pig nose at higher levels and transmit more rapidly to other pigs. This study demonstrated how the evolution and exchange of gene segments in pig populations alters IAV transmission and that these events can provide an explanation for why genetically related viruses with different gene combinations experience rapid fluxes in detection frequency. These data improve researchers⿿ ability to identify strains to include in vaccines that have gene combinations with biological properties that impact transmission and replication within pig populations. 02 Inoculated pigs with highly pathogenic avian influenza (HPAI) H5N1 viruses. HPAI have potential to cross species barriers and cause pandemics in humans. Since 2022, H5N1 HPAI has infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing mammalian adaptation mutations increase the risk of infection and subsequent exchange of viral genes with influenza A viruses (IAV) endemic in swine. This exchange of genes increases the risk of transmission within and between swine holdings. ARS scientists in Ames, Iowa, assessed the susceptibility of swine to avian and mammalian H5N1 HPAI strains using a pathogenesis and transmission model. All strains replicated in the lower respiratory tract of pigs and caused lesions in the respiratory tract consistent with IAV. Increased viral replication in the nasal cavity, transmission to contact pigs, lesions in the lung at the cellular level, and viral antigen distribution within the lung suggested an advantage in swine for strains isolated from mammals. Mammalian adaptation and reassortment may increase the risk of incursion and transmission of HPAI in feral, backyard, or commercial swine. These results inform animal and human health of the potential for HPAI to emerge in swine. 03 A mathematical approach to identify new combinations of gene segments in influenza A viruses. New combinations of influenza A virus (IAV) genes derived from human-, swine-, or avian-origin IAVs affect virus transmission and pathology. Viruses with new combinations of genes may be undergoing rapid changes in genetic diversity that reduce the efficacy of vaccine control methods and can pose a greater risk to humans for zoonotic infection. ARS scientists in Ames, Iowa, in collaboration with computer scientists at the Iowa State University, developed algorithms that measure the distance between two evolutionary trees derived from the sequences of the virus. The software can identify the differences between the two trees as a cost score. The cost score can be used to merge gene trees together into a larger network describing how gene exchange has impacted the evolution of the virus. The algorithm can objectively identify when the genetic components of a virus are derived from different evolutionary origins and can identify when specific clusters of genes are more frequently paired together. By inference, it can be used to tell when viruses are significantly divergent. Thus, these analyses can be applied to identify genetically novel swine IAV strains for characterization, for use in vaccine development, and it may be used to search for genetic markers associated with the transmission and persistence of IAV in swine populations. 04 A machine learning software program to identify and classify influenza A viruses. Diagnostic laboratories routinely generate large genetic sequence datasets with tens or hundreds of thousands of pathogen genes and strains. Approaches to the classification of the sequences can be a time-consuming process involving curated reference data and a subject- area expert to interpret phylogenetic trees. ARS scientists in Ames, Iowa, in collaboration with scientists at the Iowa State University and Duke-NUS Medical School, developed a simple and intuitive machine learning software program, classLog, to overcome this challenge. The software automatically builds a prediction model that can be applied to classify ⿿unknown⿿ sequence data. The prediction model is portable, does not need to be retrained when new sequence data are generated, and requires no specialized training to implement. Together, these advancements reduce the time and computational needs required to identify the classification of an infectious agent based on genetic sequence data. The classLog algorithm was validated with H1 influenza A virus in swine sequences and a porcine reproductive and respiratory syndrome virus dataset and achieved near perfect accuracy with real data. The software has been integrated in quarterly reports for the USDA Influenza A virus in swine surveillance system and deployed within regional diagnostic laboratory analytical platforms. The classLog software increases researchers⿿ ability to classify endemic circulating viruses and speed response efforts by helping diagnosticians rapidly identify new viral variants. 05 The evolution of H1 influenza A viruses in swine that can be transmitted to humans resulted in a gap in pandemic preparedness. Human H1 influenza A viruses (IAV) spread to pigs in North America associated with the 1918 pandemic and more recently in the 2000s. These cross- species events led to sustained circulation of two major groups of H1 viruses in swine and increased IAV diversity in pig populations. The evolution in swine of H1 IAV led to a reduced similarity with human seasonal H1 and the vaccine strains used to protect human populations. ARS scientists in Ames, Iowa, in collaboration with investigators at Emory University and the University of Pittsburgh, quantified the genetic diversity of H1 in swine, selected representative viruses, and measured the antigenic phenotypes of swine IAV using laboratory assays and transmission experiments in ferrets. They demonstrated that some North American swine H1 lineages were significantly different from historical and recent human vaccine strains. Additionally, pandemic preparedness vaccine strains developed for public health demonstrated a loss in similarity with contemporary swine strains. Lastly, one of the swine IAV lineages was able to transmit from ferret-to-ferret even in the context of vaccination with human seasonal H1. These results suggest gaps in current pandemic preparedness efforts and this information can help guide candidate vaccine development for public health. 06 Biomarkers to differentiate vaccine-associated enhanced respiratory disease (VAERD) from uncomplicated influenza A virus (IAV) infection. Previous research studies have demonstrated IAV whole-inactivated virus (WIV) vaccines in swine may lead to VAERD after heterologous viral infection. The significance of VAERD in swine production systems is unknown, in part due to lack of established clinical parameters or biomarkers for diagnosis. ARS scientists in Ames, Iowa, in collaboration with scientists at Iowa State University, evaluated multiple biomarkers in swine blood and bronchoalveolar lavage fluid to identify those that differentiated VAERD from uncomplicated influenza A infection. VAERD was associated with an increase in white blood cells and neutrophils and the acute phase proteins c-reactive protein and haptoglobin, and the cytokine IL-8 in bronchoalveolar lavage fluid compared to non-vaccinated infected pigs. These data provide an initial clinical diagnostic assessment of potential biomarkers for field diagnosis of VAERD to enable producers, veterinarians, and diagnosticians to identify the potential undesirable effects of WIV usage. 07 An introduction of human seasonal H3N2 influenza A virus was detected in swine. Multiple human-to-swine spillover events of H3N2 influenza A virus (IAV) were detected in the 2022-2023 human influenza season. These detections occurred in seven states: Colorado, Illinois, Indiana, Missouri, North Carolina, Ohio, and Pennsylvania as well as the countries of Chile and Mexico. ARS scientists in Ames, Iowa, in collaboration with scientists at Iowa State University Veterinary Diagnostic Laboratory conducted genetic and antigenic analyses to assess whether the spillover was persisting in pigs. Next generation sequencing generated whole genome sequences and were used to demonstrate that the human viruses collected in pigs were cocirculating on farms that had endemic swine IAV. The viruses were antigenically distinct from endemic swine IAV virus but were still antigenically similar to H3 viruses that were circulating in humans between 2020 and 2023. Pigs experimentally inoculated with a representative isolate exhibited minimal to mild macroscopic lung lesions, and swine-to-swine transmission was not observed. These strains of H3 represent an emerging threat to swine health through the introduction of new viral diversity and should be monitored by producers, veterinarians, and diagnosticians to assess whether they evolve to become more transmissible in the swine host. 08 The role of a host protease gene in susceptibility to influenza A virus infection. Influenza A virus (IAV) infection is initiated by hemagglutinin (HA), a glycoprotein exposed on the virion lipid envelope that undergoes cleavage by host cell proteases to ensure membrane fusion, entry into the host cells, and completion of the viral cycle. Transmembrane protease serine S1 member 2 (TMPRSS2), a host transmembrane protease, is expressed throughout the porcine airway epithelium and is purported to play a major role in the HA cleavage process, thereby influencing viral pathogenicity and tissue tropism. ARS scientists in Ames, Iowa, in collaboration with scientists at the University of Missouri, tested whether knocking out expression of TMPRSS2 will reduce IAV infectivity in the swine model. Pathogenesis of an H1N1pdm09 virus challenge in wildtype (WT) control and in TMPRSS2 knockout (KO) pigs was compared . No differences in nasal viral shedding and lung lavage viral titers were observed between the WT and KO animals. However, the KO animal group had significantly less lung lesions and significant reductions in antiviral and proinflammatory cytokines in the lung. TMPRSS2 expression was dynamic in WT pigs and modulated by IAV infection. The virus titer results in our direct challenge model contradict prior studies performed on the murine animal model, but the reduced lung lesions and cytokine profile suggest a possible role for TMPRSS2 in the proinflammatory antiviral response. These data indicate there is a need for geneticists and influenza scientists to further investigate the role of TMPRSS2 in swine IAV infection and disease. 09 Characterized endemic circulation of influenza A virus (IAV) in farms with vaccinated sows. IAV vaccines have been routinely used in sows to control disease on farms. Despite that, virus is still detected circulating in suckling and nursery pigs, allowing its dissemination within and between farms. In this study, ARS scientists in Ames, Iowa, in collaboration with scientists at Iowa State University, evaluated the presence of IAV in suckling and nursery piglets from IAV-vaccinated sows with history of influenza infection. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. Influenza virus was detected in approximately 1.64% and 31% of nasal swabs and oral fluids, respectively. An H1N2 was detected most often with sporadic detection of H1N1 and H3N2. Whole genome sequences of IAV isolated from suckling piglets revealed an H1N2 circulating in this age, and a similar H1N2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nursery from farms with endemic infections, despite using farm-specific IAV vaccines in sows. These data inform producers, veterinarians, and vaccine manufacturers on the impact of vaccine use in sows. 10 Quantified the global genetic diversity of Porcine reproductive and respiratory syndrome virus and developed a classification framework to name different groups of viruses circulating in pigs. Understanding how Porcine reproductive and respiratory syndrome virus (PRRSV) are changing in swine in a timely manner is critical to reduce disease burden in pigs and identify when novel viruses are spreading through swine populations. The genetic makeup of PRRSV continually changes, yet there are no established methods to report significant genetic changes in the regularly sequenced viral surface protein gene (ORF5). ARS scientists in Ames, Iowa, in collaboration with scientists at the Iowa State University and University of Minnesota Veterinary Diagnostic Laboratories analyzed a large dataset of swine ORF5 genetic sequences and developed an adaptable naming system that uses measurable criteria to define historical and contemporary evolutionary patterns. Using this system, 82,237 sequences collected between 1989 and 2021 from around the globe were classified. These data revealed continual turnover of different PRRSV groups, with eleven lineages and 21 sublineages within these groups that were necessary to define observed PRRSV genetic diversity. This study refined PRRSV-2 ORF5-based phylogenetic classification and demonstrated how diversity varied geographically and temporally. The refined classification system and the molecular epidemiology data in this study will aid and improve our ability to control PRRSV through identifying field-relevant virus strains and spatial and temporal changes in major groups of ORF5 genes circulating in pigs.

Impacts
(N/A)

Publications

Zeller, M.A., Arendsee, Z.W., Smith, G.J., Anderson, T.K. 2023. classLog: Logistic regression for the classification of genetic sequences. Frontiers in Virology. https://doi.org/10.3389/fviro.2023.1215012.
Poeta Silva, A.S., Almeida, M., Michael, A., Rahe, M.C., Siepker, C., Magstadt, D.R., Pineyro, P., Arruda, B.L., Macedo, N.R., Sahin, O., Gauger, P., Krueger, K., Mugabi, R., Streauslin, J.S., Trevisan, G., Linhares, D., Silva, G.S., Fano, E., Main, R., Schwartz, K.J., Burrough, E.R., Derscheid, R.J., Sitthicharoenchai, P., Clavijo, M.J. 2023. Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa state university veterinary diagnostic laboratory. BMC Veterinary Research. https://doi.org/10.1186/s12917-023-03807-w.
Yim-Im, W., Anderson, T.K., Paploski, I.A., Vanderwaal, K., Gauger, P.C., Krueger, K., Shi, M., Main, R., Zhang, J. 2023. Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes. Microbiology Spectrum. https://doi.org/10.1128/spectrum.02916-23.
Ciacci Zanella, G., Snyder, C.A., Arruda, B.L., Whitworth, K., Green, E., Poonooru, R.R., Telugu, B.P., Baker, A.L. 2024. Pigs lacking TMPRSS2 displayed fewer lung lesions and reduced inflammatory response when infected with influenza A virus. Frontiers in Genome Editing. https://doi. org/10.3389/fgeed.2023.1320180.
Arruda, B.L., Baker, A.L., Buckley, A.C., Anderson, T.K., Torchetti, M., Hines Bergeson, N., Killian, M.L., Lantz, K. 2024. Divergent pathogenesis and transmission of highly pathogenic avian influenza A(H5N1) in swine. Emerging Infectious Diseases. https://doi.org/10.3201/eid3004.231141.
Silva Dias, A., Baker, A.L., Baker, R.B., Zhang, J., Zeller, M.A., Kitikoon, P., Gauger, P.C. 2024. Detection and characterization of influenza A virus endemic circulation in suckling and nursery pigs originating from vaccinated farms in the same production system. Viruses. https://doi.org/10.3390/v16040626.
Wymore Brand, M.J., Souza, C.K., Gauger, P., Arruda, B.L., Baker, A.L. 2024. Biomarkers associated with vaccine-associated enhanced respiratory disease following influenza A infection in swine. Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.vetimm.2024.110787.
Zeller, M., Moraes, D., Ciacci Zanella, G., Souza, C.K., Anderson, T.K., Baker, A.L., Gauger, P.C. 2024. Reverse zoonosis of the 2022-2023 human seasonal H3N2 detected in swine. Viruses. https://doi.org/10.1038/s44298- 024-00042-4.
Le Sage, V., Rockey, N.C., French, A.J., McBride, R., McCarthy, K.R., Rigatti, L.H., Shephard, M.J., Jones, J.E., Walter, S.G., Doyle, J.D., Xu, L., Barbeau, D.J., Wang, S., Frizzell, S.A., Myerburg, M.M., Paulson, J.C., McElroy, A.K., Anderson, T.K., Baker, A.L., Lakdawala, S.S. 2024. Potential pandemic risk of circulating swine H1N2 influenza viruses. Nature. https://doi.org/10.1038/s41467-024-49117-z.
Kaplan, B.S., Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Velayudhan, B., Li, F., Vincent, A.L. 2020. Virus strain influenced the interspecies transmission of influenza D virus between calves and pigs. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.13943.
Junqueira, D.M., Tochetto, C., Anderson, T.K., Gava, D., Haach, V., Cantao, M.E., Baker, A.L., Schaefer, R. 2023. Human-to-swine introductions and onward transmission of 2009 H1N1 pandemic influenza viruses in Brazil. Frontiers in Microbiology. 14. https://doi.org/10.3389/fmicb.2023.1243567.
Cardenas, M., Seibert, B., Cowan, B., Fraiha, A.S., Carnaccini, S., Gay, C. L., Faccin, F., Caceres, J.C., Anderson, T.K., Baker, A.L., Perez, D.R., Rajao, D.S. 2024. Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs. PLoS Pathogens. https://doi.org/10.1371/journal.ppat. 1012026.
Thomas, M.N., Ciacci Zanella, G., Cowan, B., Caceres, J.C., Rajao, D.S., Perez, D.R., Gauger, P.C., Baker, A.L., Anderson, T.K. 2024. Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine. Journal of Virology. https://doi.org/10.1128/jvi.01703-23.
Wagle, S., Markin, A., Gorecki, P., Anderson, T.K., Eulenstein, O. 2024. Asymmetric cluster-based measures for comparative phylogenetics. Journal of Computational Biology. https://doi.org/10.1089/cmb.2023.0338.
Caceres, J.C., Cardenas-Garcia, S., Jain, A., Gay, C.J., Carnaccini, S., Seibert, B., Ferreri, L.M., Geiger, G., Jasinskas, A., Nakajiima, R., Rajao, D.S., Isakova-Sivak, I., Rudenko, L., Baker, A.L., Davies, D., Perez, D. 2021. Development of a novel live attenuated influenza A virus vaccine encoding the IgA-inducing protein. Vaccines. 9(7). Article 703. https://doi.org/10.3390/vaccines9070703.
Cook, P.W., Stark, T., Jones, J., Kondor, R., Zanders, N., Benfer, J., Scott, S., Jang, Y., Janas-Martindale, A., Lindstrom, S., Blanton, L., Schiltz, J., Tell, R., Griesser, R., Shult, P., Reisdorf, E., Danz, T., Fry, A., Barnes, J., Vincent, A.L., Wentworth, D.E., Davis, T. 2020. Detection and characterization of swine-origin Influenza A(H1N1) pandemic 2009 viruses in humans following zoonotic transmission. Journal of Virology. 95(2). https://doi.org/10.1128/JVI.01066-20.

Progress 10/01/22 to 09/30/23

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. In support of Objective 1, Subobjective 1.2, to conduct whole genome analyses and develop or extend existing methods to identify linked evolution within and between gene segments using phylogenetic networks, a novel algorithm was developed that determines how different clusters of genes are linked. The method allows different influenza A virus gene trees to be merged to generate a single hypothesis on the evolutionary history of the virus while accounting for reassortment. An evolutionary study on H1N1, H1N2, and H3N2 whole genome sequence data was performed to determine the preferential pairings between different gene segments based on the frequency of reassortment among the segments. In support of Objective 1, Subobjective 1.4, to characterize wildtype viruses antigenically by hemagglutination inhibition or neuraminidase inhibition assays or in vitro assays, genetically representative viruses 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 conducted with an H1N2 human variant strain. A pathogenesis and transmission study was performed with four strains of swine H1N1 of human seasonal H1N1pdm09 origin, but differing in year of spillover and/or genotypes. A pathogenesis and transmission study was performed in pigs to evaluate the risk of mammalian strains of H5N1 from the current highly pathogenic avian influenza virus outbreak in the U.S. In support of Objective 2, Subobjective 2.2, to sequence IAV through transmission chain in heterologous vaccine study and characterize mutants by antigenic assays, a study was conducted to evaluate the impact of vaccine immune escape on IAV evolution following experimental challenge. Samples were taken from pigs challenged by influenza A virus following vaccination. Sequencing using long-read technology is in progress, and data will be analyzed with a novel fast and lightweight software pipeline that performs genome assembly and identifies the emergence of influenza A virus variant populations within and between pigs. In support of Objective 2, Subobjective 2.3, to identify biomarkers for virus-host interactions, immunohistochemistry assays and machine learning image analysis were developed to quantify differential expression of Mucin-4 subunits in lungs of pigs infected with influenza A virus. In support of Objective 2, Subobjective 2.5, to test predicted antigenic targets in different vaccine platforms against influenza A virus (IAV) to represent circulating diversity and the effect of matching HA/NA vaccine components, a study was conducted to evaluate a vaccine implant and duration of immunity. A study was conducted to evaluate an H5 mRNA vaccine in pigs against a mammalian strain from the current highly pathogenic avian influenza virus outbreak. A study is in progress to evaluate the role of matched or mismatched maternal antibody and weaning stress in protection from homologous or heterologous challenge in piglets. Artificial Intelligence (AI)/Machine Learning (ML) Two non-neural machine learning methods were developed to determine the host-origin and temporal-origin of influenza A viruses (IAV) detected in swine. The first model was trained on 705 swine IAV gene segments and 12, 823 human IAV gene segments on local computing hardware, with the code shared at https://github.com/flu-crew/pdm-spillovers. The second model introduced a generalized, light-weight ML software that can be deployed on local computers or HPC clusters for the classification of genetic sequences from surveillance https://github.com/flu-crew/classLog. Both methods facilitate the analysis of large volumes of genetic sequence data derived from the national USDA influenza A virus in swine surveillance system. The generalized ML software was integrated into a regional veterinary diagnostic laboratory testing platform and acts as an early- warning system that detects when novel influenza A viruses are isolated in clinical diagnostic submissions. ACCOMPLISHMENTS 01 Mucin 4 protein expression can be used as an indicator of influenza A virus disease in lungs. Influenza A virus infects the cells lining the respiratory tract in the human and swine host, causing these cells to die (necrotizing bronchiolitis). Large glycoproteins called mucins protect these surfaces. Mucin 4 (MUC4) is a protein on the surface of the cell responsible for protection and involved in cell signaling to repress cell death and stimulate epithelial growth. ARS scientists in Ames, Iowa, used immunohistochemistry in combination with a machine learning image analysis algorithm to quantify differential expression of MUC4 in influenza A virus-infected and uninfected lung in a porcine model. MUC4 expression was significantly increased in bronchioles with necrotizing bronchiolitis. Increased expression of MUC4 is likely a regenerative response and further work may show that MUC4 serum concentrations and expression act as a proxy for disease severity. Understanding the impact of increased expression of MUC4 during influenza A virus infection or other respiratory disease will facilitate control strategies by elucidating mechanisms associated with resistance or enhanced susceptibility to IAV improving both human and animal health. 02 A software program to analyze and identify representative and novel influenza A viruses. Diagnostic laboratories routinely generate large genetic sequence datasets with tens or hundreds of thousands of pathogen genes and strains. This introduces a significant challenge for virologists and vaccine construction where only a few virus strains from the vast array of available diversity may be studied or included in a multivalent vaccine. ARS scientists in Ames, Iowa, in collaboration with computer scientists at the Iowa State University developed a software program that identifies representative viruses. They introduced a novel computational software, PARNAS, for fast and objective selection of the most representative virus strains. The software can automatically select a minimal set of representative genes from genomic surveillance data and determine how long these genes remain representative. The development of PARNAS provides computational support for pathogen genomic surveillance as it can objectively identify representative strains and identify when circulating strains have diverged from those representatives. The software has been integrated in quarterly reports for the USDA Influenza A virus in swine surveillance system, applied to select IAV in swine strains for characterization at the World Health Organization (WHO) Consultation on the Composition of Influenza Virus Vaccines, and has been adopted by biologic companies for assay and vaccine development. PARNAS can be used to design vaccines that better reflect the genetic diversity of influenza A and other viruses circulating in the field and can identify genetically novel viruses that may require characterization and revision to vaccine formulations. 03 Quantified the global genetic and antigenic diversity of influenza A virus in swine and the interplay of transmission between humans and swine. 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 2022. 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 27 genetically distinct cocirculating swine IAV groups. Sixteen human cases with IAV of swine origin were identified and linked to eight of the 27 swine genetic groups. Sixteen of the 27 characterized swine genetic groups had reduced antibody recognition by CVV or vaccine strain antisera, identifying gaps of coverage by human pandemic preparedness vaccines. These analyses demonstrated the dynamic interplay of IAV transmission between humans and swine and identified genetic groups of swine IAV that are considered by the WHO to be of interest for pandemic preparedness efforts. 04 The evolution of H1 influenza A viruses in swine that can be transmitted to humans resulted in a gap in our pandemic preparedness. Human H1 influenza A viruses (IAV) spread to pigs in North America associated with the 1918 pandemic and more recently in the 2000s. These cross-species events led to sustained circulation of two major groups of H1 viruses in swine and increased IAV diversity in pig populations. The evolution in swine of H1 IAV led to a reduced similarity with human seasonal H1 and the vaccine strains used to protect human populations. ARS scientists in Ames, Iowa, in collaboration with the Royal Veterinary College of the University of London, quantified the genetic diversity of H1 in swine, selected representative viruses, and measured the diversity of antigenic phenotypes cocirculating in North American pigs. They demonstrated that North American swine H1 lineages were significantly different from historical and recent human vaccine strains and this antigenic dissimilarity increased over time as the viruses evolved in swine. Additionally, pandemic preparedness vaccine strains developed for public health demonstrated a loss in similarity with contemporary swine strains. Lastly, post-exposure and post- vaccination human sera revealed a diversity of responses to swine IAV, including two groups of viruses where there was little to no immunity to the swine H1. Genomic surveillance and analysis paired with antigenic assessments of swine H1 IAV identified gaps in current pandemic preparedness efforts and this information can help guide candidate vaccine development for public health. 05 Identification of transmission from swine-to-ferret of swine H3 influenza A viruses is an indication of zoonotic risk to humans. Evolution of influenza A virus (IAV) in swine may result in unique viruses that pose a public health concern, and there have been a significant number of human infections from swine-origin IAV over the past decade. All circulating swine H3 subtype lineages are derived from human-to-swine interspecies transmission events and these lineages may retain human-transmissible capabilities. Contemporary H3 swine IAV exhibit significant genetic and antigenic diversity and current human seasonal vaccines or pre-pandemic candidate virus vaccines (CVV) may not protect adequately. ARS scientists in Ames, Iowa, used computational, serologic, and animal studies to perform a risk assessment of contemporary swine H3 IAV. Potential gaps in human vaccine coverage were identified and the utility of swine-to-ferret transmission experiments for risk assessment was demonstrated. Representative swine H3 viruses were efficiently transmitted from pig- to-ferret, indicating that these swine IAV represent a zoonotic risk, informing public health for pandemic preparedness. 06 Swine H1 viruses variably transmitted from pig-to-ferret, indicating that some swine H1 IAV represent relatively higher zoonotic risk. Influenza A viruses (IAV) are endemic in both humans and pigs and these viruses readily move between hosts. This interspecies transmission increases viral diversity and has great impact on viral ecology in both hosts. Swine origin IAVs have the potential to initiate human pandemics and are of great importance to pandemic preparation efforts. Because of this, swine origin IAVs have been used to generate pandemic preparedness candidate vaccine viruses (CVV), but the efficacy of these vaccines against contemporary viruses is unclear due to viral evolution. ARS scientists in Ames, Iowa, used computational, serological and in vivo studies to perform a risk assessment of contemporary swine H1 IAVs. Potential gaps in vaccine coverage were identified and the utility of swine-to-ferret transmission experiments to enhance risk assessment was demonstrated. Representative swine H1 viruses variably transmitted from pig-to-ferret, indicating that some swine H1 IAV represent relatively higher zoonotic risk, informing public health for pandemic preparedness. 07 Improved vaccines will reduce influenza disease in swine, reducing the economic and human transmission risks. Influenza A virus is a major respiratory pathogen in swine that leads to significant economic loss in the swine industry, and there is a critical need to improve on commercial vaccines. Traditional vaccines target the hemagglutinin (HA) portion of the IAV virus, lose protection as viruses change, and may even lead to vaccine-associated enhanced respiratory disease (VAERD) after infection with a dissimilar influenza virus. A newer replicon particle (RP) vaccine platform targeting the influenza HA protein offers multiple advantages over traditional vaccines for swine, but have not yet been evaluated for the ability to avoid VAERD or the use of HA along with an additional viral vaccine target, such as neuraminidase (NA). ARS scientist in Ames, Iowa, demonstrated RP vaccines containing HA and NA targets stimulated immune responses, protected from disease, and avoided VAERD following infection with a distantly related virus. This demonstrates the potential utility of RP vaccines against influenza and the importance in utilizing the NA in influenza vaccine design. Improvements of IAV vaccines like these will reduce influenza disease and economic loss in commercial swine and reduce the risk of influenza transmission to people. 08 The genetic determinants of antigenic diversity of N1 neuraminidase influenza A virus in swine help improve vaccine selection. Influenza A virus (IAV) is a common pathogen in swine and leads to significant production losses every year. There is significant diversity within the H1N1 subtype and the genetic determinants of antigenic phenotype within the N1 neuraminidase (NA) gene are uncharacterized. ARS scientists in Ames, Iowa, in collaboration with the University of Georgia, quantified how and why genetic diversity within the N1 gene changed between 1930 and 2020. Differences in immune response to representative swine N1 proteins from contemporary circulating groups of viruses were measured. Each of the swine N1 clades was unique, and the size of the difference was linked to the genetic distance between the viruses. N1 groups and N1 pairings with the hemagglutinin (HA) surface protein gene increased and decreased in detection frequency across North America. In rare cases, when N1 genes acquired a new HA protein through reassortment, new N1 genetic groups would emerge. This study demonstrated how influenza A virus reassortment affected the genetic and antigenic diversity of N1. These data identify emerging and dominant N1 groups and understand how genetic diversity drives the antigenic diversity of IAV in swine to improve vaccine control efforts for the swine industry. 09 The diversity and evolution of influenza A virus (IAV) in pigs is linked to the emergence of IAV with zoonotic potential. Human-to-swine transmission of the 2009 H1N1 pandemic (pdm09) IAV lineage repeatedly occurred across the past decade and has increased genetic diversity in pigs: sporadic swine-to-human cases are associated with these viruses. ARS scientists in Ames, Iowa, in collaboration with the Iowa State University Veterinary Diagnostic Laboratory measured the frequency of human-to-swine transmission of the H1N1 pandemic IAV lineage between 2009 and 2021 and determined how this affected the diversity of IAV in swine and zoonotic risk. They detected 370 separate human-to-swine spillovers, with the frequency of interspecies transmission increasing when the burden of IAV was highest in the human population. Most spillovers were single events without sustained transmission, but a small subset resulted in the emergence, persistence, and cocirculation of different pdm09 genetic clades in U.S. pigs. Each of the pdm09 representative of different persistent spillovers was genetically and antigenically different from human seasonal vaccine strains. The persistence of pdm09 within pigs resulted in at least five recent swine- to-human transmission events. These data suggest that the swine industry could reduce spillover of IAV into pigs from humans working with swine, reducing the resulting genetic diversity of IAV in pigs, and proactively reducing the potential for future swine-to-human transmission of IAV with pandemic potential.

Impacts
(N/A)

Publications

Arendsee, Z.W., Baker, A.L., Anderson, T.K. 2022. smot: a python package and CLI tool for contextual phylogenetic subsampling. Journal of Open Source Software. 7(80). Article 4193. https://doi.org/10.21105/joss.04193.
Anderson, T.K., Baker, A.L. 2022. Swine influenza A viruses and pandemic preparedness. Council for Agricultural Science and Technology Issue Paper. SP33:26-28.
Venkatesh, D., Anderson, T.K., Chang, J., Lopes, S., Kimble, B., Souza, C. K., Pekosz, A., Rothman, R.E., Chen, K., Baker, A.L., Lewis, N.S. 2022. Antigenic characterization and pandemic risk assessment of North American H1 influenza A viruses circulating in swine. Microbiology Spectrum. 10(6). Article e01781-22. https://doi.org/10.1128/spectrum.01781-22.
Arruda, B.L., Falkenberg, S.M., Mora-Diaz, J., Matias Ferreyra, F.S., Magtoto, R., Gimenez-Lirola, L. 2022. Development and evaluation of antigen-specific dual matrix Pestivirus K ELISAs using longitudinal known infectious status samples. Journal of Clinical Microbiology. 60(11). Article e00697-22. https://doi.org/10.1128/jcm.00697-22.
Rajoa, D.S., Zanella, G.C., Brand, M.W., Khan, S., Miller, M.E., Ferreri, L.M., Caceres, C., Cadernas-Garcia, S., Souza, C.K., Anderson, T.K., Gauger, P.C., Baker, A.L., Perez, D.R. 2023. Live attenuated influenza A virus vaccine expressing an IgA-inducing protein protects pigs against replication and transmission.. Frontiers in Virology. 3. Article 1042724. https://doi.org/10.3389/fviro.2023.1042724.
Kimble, J., Souza, C.K., Anderson, T.K., Arendsee, Z.W., Hufnagel, D.E., Young, K.M., Lewis, N.S., Davis, C., Thor, S., Baker, A.L. 2022. Interspecies transmission from pigs to ferrets of antigenically distinct swine H1 Influenza A viruses with reduced reactivity to candidate vaccine virus antisera as measures of relative zoonotic risk. Viruses. 14(11). Article 2398. https://doi.org/10.3390/v14112398.
Moraes, D.C., Baker, A.L., Wang, X., Zhu, Z., Berg, E., Trevisan, G., Zhang, J., Jayaraman, S., Linhares, D., Gauger, P.C., Silva, G.S. 2023. Veterinarian perceptions and practices in prevention and control of influenza virus in the midwest United States swine farms. Frontiers in Veterinary Infectious Diseases. 10. https://doi.org/10.3389/fvets.2023. 1089132.
Souza, C.K., Kimble, J.B., Anderson, T.K., Arendsee, Z.W., Hufnagel, D.E., Young, K.M., Gauger, P.C., Lewis, N.S., Davis, C.T., Sharmi, T., Baker, A. L. 2023. Swine-to-ferret transmission of antigenically drifted contemporary swine H3N2 influenza A virus is an indicator of zoonotic risk to humans. Viruses. 15(2). Article 331. https://doi.org/10.3390/v15020331.
Hufnagel, D.E., Young, K.M., Arendsee, Z., Gay, L.C., Caceres, C.J., Rajao, D., Perez, D.R., Baker, A.L., Anderson, T.K. 2023. Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in influenza A virus from North American swine. Virus Evolution. 9(1). Article 10.1093. https://doi.org/10.1093/ve/vead015.
Tochetto, C., Junqueira, D.M., Anderson, T.K., Gava, D., Haach, V., Cantao, M.E., Baker, A.L., Schaefer, R. 2023. Introductions of human-origin seasonal H3N2, H1N2, and pre-2009 H1N1 influenza viruses to swine in Brazil. Viruses. 15(2). Article 576. https://doi.org/10.3390/v15020576.
Rajao, D., Abente, E.J., Powell, J.D., Bolton, M.J., Gauger, P.C., Arruda, B.L., Anderson, T.K., Sutton, T., Perez, D.R., Baker, A.L. 2022. Changes in the hemagglutinin and internal gene segments were needed for human seasonal H3 influenza A virus to efficiently infect and replicate in swine. Pathogens. 11(9). Article 967. https://doi.org/10.3390/pathogens11090967.
Zeller, M.A., Saxena, A., Anderson, T.K., Baker, A.L., Gauger, P.C. 2022. Use of the ISU FLUture multisequence identity tool for rapid interpretation of swine influenza A virus sequences in the United States. Journal of Veterinary Diagnostic Investigation. 34(5):874-878. https://doi. org/10.1177/10406387221111128.
Mo, J., Abente, E.J., Sutton, T.C., Ferreri, L.M., Geiger, G., Gauger, P.C. , Perez, D.R., Baker, A.L., Rajao, D.S. 2022. Transmission of human influenza A virus in pigs selects for adaptive mutations on the HA gene. Journal of Virology. 96(22). Article e01480-22. https://doi.org/10.1128/ jvi.01480-22.
Arruda, B.L., Kanefsky, R.A., Hau, S.J., Janzen, G.M., Anderson, T.K., Baker, A.L. 2023. Mucin 4 is a cellular biomarker of necrotizing bronchiolitis in influenza A virus infection. Microbes and Infection. e105169. https://doi.org/10.1016/j.micinf.2023.105169.
Markin, A., Wagle, S., Grover, S., Baker, A.L., Eulenstein, O., Anderson, T.K. 2023. PARNAS: Objectively selecting the most representative taxa on a phylogeny. Systematic Biology. esyad028. https://doi.org/10.1093/sysbio/ syad028.
Wagle, S., Markin, A., Gorecki, P., Anderson, T.K., Eulenstein, O. 2023. The asymmetric cluster affinity cost. Research Computational Molecular Biology (RECOMB). 13883:131-145. https://doi.org/10.1007/978-3-031-36911- 7_9.
Markin, A., Ciacci Zanella, G., Arendsee, Z.W., Zhang, J., Krueger, K.M., Gauger, P.C., Baker, A.L., Anderson, T.K. 2023. Reverse-zoonoses of 2009 H1N1 pandemic influenza A viruses and evolution in United States swine results in viruses with zoonotic potential. PLoS Pathogens. 19(7). Article e1011476. https://doi.org/10.1371/journal.ppat.1011476.
Grover, S., Markin, A., Anderson, T.K., Eulenstein, O. 2023. Phylogenetic diversity statistics for all clades in a phylogeny. Bioinformatics. 39(1) :i177-i184. https://doi.org/10.1093/bioinformatics/btad263.
Wymore Brand, M., Anderson, T.K., Kitikoon, P., Kimble, B.J., Otis, N.J., Gauger, P.C., Souza, C.K., Kaplan, B.S., Mogler, M., Strait, E., Baker, A. L. 2022. Bivalent hemagglutinin and neuraminidase influenza replicon particle vaccines protect pigs against influenza a virus without causing vaccine associated enhanced respiratory disease. Vaccine. 40(38): 5569- 5578. https://doi.org/10.1016/j.vaccine.2022.07.042.

Progress 10/01/21 to 09/30/22

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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. 09 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.

Impacts
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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.