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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Research Project #432024

Research Project: Intervention Strategies to Control Influenza A Virus Infection in Swine

Location: Virus and Prion Research

2017 Annual Report

Objective 1. Identify mechanisms of influenza A virus (IAV) pathogenesis and host adaptation to swine. This includes investigating host-pathogen interactions at cellular or molecular levels, identifying determinants of swine IAV infection and shedding from respiratory mucosa, and investigating host range restriction to identify mechanisms by which non-swine adapted viruses infect and adapt to swine. Objective 2. Evaluate emerging IAV at the genetic and antigenic levels as a risk to swine or other host species. This includes identifying emerging IAV and monitoring genetic and antigenic evolution in swine, and identifying genetic changes important for antigenic drift or pathogenicity in swine or other hosts. Objective 3. Identify novel influenza vaccine platforms and improve vaccination strategies. This includes characterizing humoral and cellular immune responses to wild-type and attenuated viruses compared to inactivated vaccines to identify correlates of protection, investigating adjuvants or immune-modulatory agents that result in robust immune responses (mucosal delivered, long lived, broadly cross-protective and/or reduce the number of vaccine boosters), and investigating technologies to override IAV vaccine interference from passively acquired immunity.

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

Progress Report
In support of Objective 1, Subobjective 1.1, to investigate host-pathogen interactions at cellular or molecular levels, RNA was extracted from porcine alveolar macrophages and lung tissue from pigs infected with influenza A virus (IAV) from a previously completed study. Host gene expression profiles were examined using a PCR array targeting 168 genes associated with the swine antiviral response and cytokine and chemokine pathways. Differential gene expression patterns were observed. In support of Objective 1, Subobjective 1.3, to examine virus, host, and population factors that influence interspecies transmission in swine, work continued on a recently established human-like H3 virus lineage in swine to study its genetic and antigenic evolution. Representative human and swine human-like viruses were used to perform virus histochemistry on swine tissue and in vitro replication assays. A pathogenesis and transmission study with a North American 2017 H7N9 low pathogenic avian influenza virus was completed. In support of Objective 2, Subobjective 2.1a, to identify emerging IAV and monitor genetic and antigenic evolution in swine, subtype and genetic patterns were monitored to identify changing patterns or emerging viruses. H1N1, H1N2, and H3N2 with molecular signatures suggesting antigenic changes were identified and virus isolates obtained from the USDA IAV-S surveillance repository for antigenic and pathogenic characterization. In support of Objective 2, Subobjective 2.1b, to develop and implement an automated clade tool for H1 with standardized global nomenclature, a phylogenetic based method for classifying H1 IAV was developed and validated on a large global dataset of hemagglutinin gene sequences. The automated tool was demonstrated to be highly accurate and was implemented on the Influenza Research Database ( In support of Objective 2, Subobjective 2.2, to identify genetic changes important for antigenic drift or pathogenicity in swine or other hosts, IAV subtype H1 and H3 viruses with unique antigenic motifs, predicted to be antigenically distinct, were obtained and tested in vitro to characterize their antigenic phenotypes. New antigenic motif patterns in H3 were shown to be distinct from previous H3 and changed in frequency of detection over time. In support of Objective 3, Subobjective 3.1, to characterize humoral and cellular immune responses to wild-type and attenuated viruses compared to inactivated or vectored vaccines to identify correlates of protection, a study was completed to compare whole inactivated virus (WIV), live attenuated influenza virus (LAIV), and an RNA vectored vaccine platform against IAV with H3 hemagglutinins that differed in only a few key amino acid positions. The LAIV and RNA vectored vaccines demonstrated superior protection from heterologous challenge. Another study to compare WIV and RNA vectored vaccine was conducted with H1 viruses. In support of Objective 3, Subobjective 3.2, to investigate adjuvants or immune-modulatory agents that result in robust immune responses (mucosal delivered, long lived, broadly cross-protective, and/or reduce the number of vaccine boosters), a study was conducted to test the effect of sequential heterologous infection in imprinting the humoral immune response. The order of infection significantly impacted the humoral immune response to each of the viruses and certain exposure patterns led to increased lung pathology.

1. Developed a computational tool that automatically classifies global swine H1 subtype HA gene sequences. Infection with influenza A virus (IAV) is one of the most important respiratory diseases of swine and is the second most common viral diagnosis of respiratory disease in the United States. The USDA IAV swine surveillance system initiated in 2009 has increased the amount of publically available sequence data on swine viruses circulating in the United States. A significant barrier for swine producers to make timely vaccine interventions and for researchers to use relevant viruses in studies is having the computational expertise to analyze and characterize the HA gene. The HA protein is a major component of vaccines and target for immune responses. In collaboration with an international network of influenza experts, ARS researchers at Ames, Iowa developed a computational tool that can automatically classify swine H1 subtype HA gene sequences. An important component of the tool is the harmonization of H1 HA nomenclature, as well as a standardized technique for genetically characterizing the HA gene. This open-access tool will aid swine producers, veterinarians, vaccine manufacturers, and IAV vaccine researchers in selecting vaccine strains to match the strains that are currently circulating. Properly matching vaccines to field strains is a critical part of managing swine influenza.

2. Reassortant influenza A virus (IAV) with highly pathogenic avian influenza H5N1 surface genes had modestly increased replication and transmission in pigs. Following the introduction of the 2009 pandemic H1N1 virus (H1N1pdm09), many animal species have been shown to be infected due to human to animal transmission. The IAV genome is composed of 8 gene segments, and mixing of gene segments from distinct parental viruses can result in progeny viruses with improved capability of infecting a host, ability to evade immunity, or with distinct pathogenic phenotypes. ARS scientists in Ames, Iowa demonstrated that a laboratory generated reassortant virus with highly pathogenic avian influenza H5N1 surface genes and internal genes from H1N1pdm09 virus had modestly increased replication and transmission in pigs when compared to the parental H5N1 virus. Although not yet detected in pigs from natural events, this finding highlights the importance of maintaining a robust surveillance program to detect spillover events into swine and suggests that interspecies transmission barriers may partially be overcome by reassortment. Interspecies transmission into pigs is a risk to swine production as well as human pandemic risk.

3. Demonstrated properties of H3N2 influenza A virus (IAV) strains isolated from swine varied depending on the genome constellation. Following the introduction of the 2009 pandemic H1N1 (H1N1pdm09) from humans to swine, mixing of IAV gene segments between H1N1pdm09 and swine viruses occurred. By studying genomes of IAV detected in swine, a large number of gene segment combinations (genomes) among H3 subtype swine viruses were shown to be circulating in commercial herds. ARS researchers at Ames, Iowa selected IAV with genomes representing observed patterns in viruses circulating in swine farms to investigate in experimental challenge studies. Infection properties of viral strains varied depending on the genome constellation and may explain why some combination of genes have been more successful in the U.S. swine population. This underscores the importance of surveillance and assessing whole-genome sequence data to better understand the disease properties of circulating IAV strains in the field. This information will help guide intervention strategies and improved choices in vaccine design.

4. Demonstrated pigs with severe combined immunodeficiency (SCID) were impaired in controlling influenza A virus (IAV) infection. Influenza A virus infections tend to be acute and relatively short in duration due to rapid induction of the immune response. Study of the immune response to IAV can reveal new ways to prevent or treat infections. Humans and animals may have genetic disorders that interrupt normal immune responses. In collaboration with scientists at Iowa State University, ARS researchers at Ames, Iowa showed that pigs with SCID that do not have B-cell or T-cell immunity were impaired in controlling IAV infection. The delayed clearance of infection was despite an intact innate immune response. These SCID pigs provide a valuable model to understand the immune mechanisms associated with protection and recovery in a natural host for influenza.

5. Mammals captured near infected poultry farms lack evidence of exposure to 2014-2015 highly pathogenic avian influenza virus. In 2014 and early 2015, a Eurasian strain of highly pathogenic avian influenza A (HPAI) virus was detected in poultry in Canada and the United States, causing a large economic loss to the poultry industry and tremendous investment by the industry and USDA officials to control the outbreak. In an effort to understand the spread of the Eurasian H5 virus, epidemiologic investigations occurred at poultry facilities. Synanthropic birds and mammals were sampled at infected and uninfected poultry farms in northwest Iowa, and in collaboration with APHIS scientists, ARS researchers at Ames, Iowa tested for evidence of infection with HPAI H5. No mammal species showed evidence of infection or exposure, but a very small number of European starlings were found to have evidence of infection. These results indicate species that cohabitate with humans and their domestic animals merit further scrutiny to better understand potential biosecurity risks to HPAI outbreaks.

Review Publications
Abente, E.J., Kitikoon, P., Lager, K.M., Gauger, P.C., Anderson, T.K., Vincent, A.L. 2017. A highly pathogenic avian-derived influenza virus H5N1 with 2009 pandemic H1N1 internal genes demonstrates increased replication and transmission in pigs. Journal of General Virology. 98(1):18-30.
Anderson, T.K., Macken, C.A., Lewis, N.S., Scheuermann, R.H., Van Reeth, K., Brown, I.H., Swenson, S.L., Simon, G., Saito, T., Berhane, Y., Ciacci-Zanella, J., Pereda, A., Davis, C.T., Donis, R.O., Webby, R.J., Vincent, A.L. 2016. A phylogeny-based global nomenclature system and automated annotation tool for H1 hemagglutinin genes from swine influenza A viruses. mSphere. 1(6):e00275-16. doi:10.1128/mSphere.00275-16.
Olson, Z.F., Sandbulte, M.R., Kunzler Souza, C., Perez, D.R., Vincent, A.L., Loving, C.L. 2017. Factors affecting induction of peripheral IFN-gamma recall response to influenza A virus vaccination in pigs. Veterinary Immunology and Immunopathology. 185:57-65. doi: 10.1016/j.vetimm.2017.01.009.
Rajao, D.S., Loving, C.L., Waide, E.H., Gauger, P.C., Dekkers, J.C., Tuggle, C.K., Vincent, A.L. 2017. Pigs with severe combined immunodeficiency are impaired in controlling influenza virus infection. Journal of Innate Immunity. 9(2):193-202.
Rajao, D.S., Walia, R.R., Campbell, B., Gauger, P.C., Janas-Martindale, A., Killian, M.L., Vincent, A.L. 2017. Reassortment between swine H3N2 and 2009 pandemic H1N1 in the United States resulted in influenza A viruses with diverse genetic constellations with variable virulence in pigs. Journal of Virology. 91:e01763-16.
Sandbulte, M.R., Gauger, P.C., Kitikoon, P., Chen, H., Perez, D.P., Roth, J.A., Vincent, A.L. 2016. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type. Vaccine. 34(33):3773-3779.
Vincent, A.L., Perez, D.R., Rajao, D., Anderson, T.K., Abente, E.J., Walia, R.R., Lewis, N.S. 2016. Influenza A virus vaccines for swine. Veterinary Microbiology. 206:35-44.
Zhang, Y., Aevermann, B., Anderson, T.K., Burke, D.F., Dauphin, G., Gu, Z., He, S., Kumar, S., Larsen, C.N., Lee, A.J., Li, X., Macken, C., Mahaffey, C., Pickett, B.E., Reardon, B., Smith, T., Stewart, L., Suloway, C., Sun, G., Tong, L., Vincent, A.L., Walters, B., Zaremba, S., Zhao, H., Zhou, L., Zmasek, C., Klem, E.B., Scheuermann, R.H. 2017. Influenza research database: an integrated bioinformatics resource for influenza virus research. Nucleic Acids Research. 45(D1):D466-D474. doi:10.1093/nar/gkw857.
Shriner, S.A., Root, J.J., Lutman, M.W., Kloft, J.M., VanDalen, K.K., Sullivan, H.J., White, T.S., Milleson, M.P., Hairston, J.L., Chandler, S.C., Wolf, P.C., Turnage, C.T., McCluskey, B.J., Vincent, A.L., Torchetti, M.K., Gidlewski, T., DeLiberto, T.J. 2016. Surveillance for highly pathogenic H5 avian influenza virus in synanthropic wildlife associated with poultry farms during an acute outbreak. Scientific Reports. 6:36237. doi: 10.1038/srep36237.