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

Location: Virus and Prion Research

2023 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 1.2, 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.

Accomplishments
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

Review 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.
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.
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.