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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

2018 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 1.2, to develop in vitro assays to predict infection, replication, and/or shedding in swine, a swine cell line that is preferentially infected by swine influenza A virus (IAV) was found to correlate with susceptibility in pigs from animal studies. Additional swine and non-swine IAV were tested to assess the potential use of this cell line to predict susceptibility and virus infection phenotype in pigs. In support of Objective 1, Subobjective 1.3, to identify HA or NA or polymerase complex factors for adaptation in swine versus non-swine adapted IAV in vitro, reverse-genetics was employed to generate reassortant and HA mutant viruses to identify molecular determinants for adaptation of non-swine viruses. In support of Objective 2, Subobjective 2.1a, to correlate rates of evolution with persistence of genotypes in the population of circulating viruses, phylogenetic methods were used to infer molecular evolutionary rates of hemagglutinin gene sequences. The most dominant viruses based upon surveillance data demonstrated increased evolutionary rates, and structural modeling of the hemagglutinin suggested these evolutionary dynamics cause antigenic drift. In support of Objective 2, Subobjective 2.1b, to develop and implement an automated clade tool for the H3 IAV subtype with standardized global nomenclature, an annotation tool and statistical criteria were developed. We analyzed more than 210,000 H3 HA sequences collected from 1969 to 2017 and were able to annotate appropriate evolutionary names to swine and other host H3 HA sequences correctly more than 95% of the time: this tool will be implemented on the Influenza Research Database. In support of Objective 2, Subobjective 2.2, to test amino acid substitutions in H3 for antigenic evolution, a broad panel of contemporary H3 viruses with uncharacterized antigenic motifs were selected across multiple clades within the phlylogenetic clade called Cluster-IV (C-IV) to assess the impact of overall HA diversity on the antigenic phenotype.

1. A novel subtype of influenza A virus (IAV) detected in pigs in the U.S.A. There are three subtypes of IAV found in swine globally: H1N1, H1N2 and H3N2. In addition to pigs, IAV is an important pathogen in humans, birds and other host species, and infection with viruses between hosts plays an important role in the evolution of this virus. IAV strains that are adapted to one type of host sporadically infect different host species and may lead to an outbreak in the new host population. In December of 2015 a subtype of IAV not normally found in pigs but common in wild ducks, H4N6, was detected in sick pigs. In an experimental challenge study conducted by ARS scientists in Ames, Iowa, this H4N6 virus showed limited ability to infect the lungs of pigs, no virus was detected in the nasal cavity, and there was no evidence of spread from pig-to-pig. The experimental results suggest the H4N6 was not likely to be successful in pigs and follow up testing on the originating pig farm indicated that the avian H4N6 was not sustained and quickly died out. Interspecies transmission into pigs is a risk to swine production as well as a human pandemic risk.

2. An emerging lineage of influenza A virus (IAV) in the U.S. swine population caused outbreaks of human infection associated with agricultural fairs. IAV in pigs impacts swine health through reduced production and costs, but also represents a public health concern as human immunity to these swine IAV may be reduced and there is a risk for spillover to humans. When humans are infected with a swine IAV, it is termed "variant" to differentiate it from typical human seasonal strains. In 2016, 18 cases of variant A/H3N2 IAV were detected in people following their attendance at swine exhibits at agricultural fairs. Samples collected from swine from the same location and during the same window of time as the human infections provided clear molecular evidence of zoonotic IAV transmission from the pigs to humans. ARS scientists in Ames, Iowa, provided evidence that the genetic sequences of the viruses isolated from the pigs were nearly identical to viruses identified in the humans. These results demonstrate the need for continued genetic and antigenic characterization of swine IAV to determine potential vaccine and intervention efforts that minimize transmission in swine herds and to exhibited swine and from exhibited swine to human fair attendees. This outbreak emphasizes a "One Health" approach between public health and animal health sectors to investigate and respond to variant IAV outbreaks.

3. The 2009 H1N1 pandemic virus caused dramatic evolution of swine influenza A viruses (IAV). The 2009 H1N1 swine-origin pandemic virus spread rapidly among humans, causing the first pandemic in over 40 years. Although the immediate origin of this virus was from swine, it contained genes derived from avian, human, and swine IAV lineages. The H1N1pdm09 replaced the previous human seasonal H1N1, but in US swine populations, it is detected only at low levels, mainly through repeated human-to-swine transmission. Despite seemingly low prevalence in swine, we provided evidence that the pandemic virus shared its genetic material with swine H1 IAV, generating many new genetic variants of viruses and novel gene segment combinations. The detection of different genotypes was dynamic from year to year; some of the genotypes increased in frequency, whereas others decreased or ceased to be detected. ARS scientists in Ames, Iowa demonstrated dramatic changes in swine IAV genetic diversity following 2009, which allowed for rapid genetic evolution. Detection of emerging IAV is important for developing control strategies for swine and for human pandemic preparedness.

4. Highly pathogenic avian influenza A virus (IAV) H5N1 and H5N2 from a North American poultry outbreak did not infect pigs. IAV originating in birds can infect and cause severe disease and potentially death in pigs and humans. In 2014, a highly pathogenic avian influenza (HPAI) H5N8 was introduced to North America by migrating waterfowl. Introduction of H5N8 allowed swapping of its genetic material with other avian strains from North America, resulting in two additional subtypes, H5N1 and H5N2. The H5N2 spread across the USA, presumably by wild birds, and was isolated from poultry farms in the Midwest, resulting in millions of dollars in losses. Although there were no documented cases of H5N1, H5N2, or H5N8 in US swine, understanding the risk these viruses pose to domestic pigs is important for veterinary health, as the pig density is very high in the same region as the poultry detections. The risk of spillover of these HPAI viruses into swine is also important for human health as the ability to infect swine could lead to greater ability to infect humans following mammalian adaptation in the swine host. ARS researchers in Ames, Iowa showed that HPAI viruses of the three subtypes tested posed a low risk for infection and transmission among pigs, important information for agriculture and public health should they return again to the USA or elsewhere in the world where they circulate.

5. Influenza A viruses (IAV) that circulate in North American pigs maintain a high degree of diversity, especially those of the H1 subtype, determined by the hemagglutinin (HA) gene. The HA protein is the primary target of protective immune responses and the major consideration for vaccine formulation. The way immune antibodies recognize and bind to the HA protein affect its antigenic properties. One process of virus evolution, called antigenic drift, may lead to potential mismatches between vaccines and circulating strains. ARS researchers in Ames, Iowa found that changes in H1 from IAV in the U.S. swine population resulted in seven distinct antigenic groups of the H1 subtype. The changes in the HA protein were associated with the observed antigenic drift which may allow for prediction of antigenic changes bases on genetic sequence. The continued genetic and antigenic evolution of contemporary H1 viruses may lead to loss of vaccine protection, with significant economic impact to the swine industry and represents a challenge to public health initiatives to minimize human exposure to IAV from swine.

6. A new type of influenza A virus (IAV) vaccine was tested in swine. IAV in swine constitutes a major economic burden for producers. Current IAV vaccines used in swine are often marginally effective at preventing disease and do not provide cross-protection against the diversity of IAV co-circulating in pig herds in the United States. ARS researchers in Ames, Iowa evaluated a live attenuated influenza vaccine (LAIV) in pigs. Pigs inoculated with the LAIV displayed no outward clinical disease or detectable lung pathology, demonstrating that the virus was attenuated in pigs. Results also show high levels of antibodies in the blood following vaccination, indicating that these modified viruses can induce a strong immune response. Vaccinated pigs were then challenged with the matching IAV and pigs were protected from infection. These results showed the LAIV virus was attenuated by the modifications and provided protection in pigs, warranting further development for use in swine. New vaccine approaches, such as the CPBD LAIV, are needed for swine to reduce the burden of influenza disease.

7. Influenza A viruses (IAV) in swine are diverse and rapidly evolve. Understanding the genetic diversity and patterns of IAV evolution in the swine population is critical for controlling this important pathogen. ARS scientists in Ames, Iowa quantified the genetic diversity of swine IAV collected from 2010-2016 at regional and national levels. Seasonal patterns of transmission of swine IAV were observed, and genetic diversity within certain regions was different than the overall national pattern. Minor variants were underreported in the global dataset, but when considered in a regional context, were important long-term components of observed diversity. The identification of these patterns demonstrates the importance of a robust surveillance system for swine IAV that includes representation from all swine production regions to inform control measures taken against IAV. Timely vaccine strain updates based that reflect regional patterns of genetic diversity may help reduce infection and transmission and improve animal health and wellbeing. This information will help guide intervention strategies and improved choices in vaccine design.

Review Publications
Abente, E.J., Gauger, P.C., Walia, R.R., Rajao, D.S., Zhang, J., Harmon, K.M., Killian, M., Vincent, A.L. 2017. Detection and characterization of an H4N6 avian-lineage influenza A virus in pigs in the midwestern United States. Virology. 511:56-65.
Martin, B.E., Sun, H., Carrel, M., Cunningham, F.L., Baroch, J.A., Hanson-Dorr, K.C., Young, S.G., Schmit, B., Nolting, J.M., Yoon, K-J., Lutman, M.W., Pedersen, K., Lager, K., Bowman, A.S., Slemons, R.D., Smith, D.R., DeLiberto, T., Wan, X-F. 2017. Feral swine in the United States have been exposed to both avian and swine influenza A viruses. Applied and Environmental Microbiology. 83(19):e01346-17.
Bowman, A.S., Walia, R.R., Nolting, J.M., Vincent, A.L., Killian, M.L., Zentkovich, M.M., Lorbach, J.N., Lauterbach, S.E., Anderson, T.K., Davis, C.T., Zanders, N., Jones, J., Yang, Y., Lynch, B., Rodriguez, M.R., Blanton, L., Lindstrom, S.E., Wentworth, D.E., Schiltz, J., Averill, J.J., Forshey, T. 2017. Influenza A (H3N2) virus in swine at agricultural fairs and transmission to humans, Michigan and Ohio, USA, 2016. Emerging Infectious Diseases. 23(9):1551-1555.
Gao, S., Anderson, T.K., Walia, R.R., Dorman, K.S., Janas-Martindale, A., Vincent, A.L. 2017. The genomic evolution of H1 influenza A viruses from swine detected in the United States between 2009 and 2016. Journal of General Virology. 98(8):2001-2010.
Kaplan, B.S., Torchetti, M.K., Lager, K.M., Webby, R.J., Vincent, A.L. 2017. Absence of clinical disease and contact transmission of HPAI H5NX clade from North America in experimentally infected pigs. Influenza and Other Respiratory Viruses. 11(5):464-470.
Rajao, D.S., Anderson, T.K., Kitikoon, P., Stratton, J., Lewis, N.S., Vincent, A.L. 2018. Antigenic and genetic evolution of contemporary swine H1 influenza viruses in the United States. Virology. 518:45-54. https://doi.10.1016/j.virol.2018.02.006.
Kaplan, B.S., Souza, C.K., Gauger, P.C., Stauft, C.B., Coleman, J.R., Mueller, S., Vincent, A.L. 2018. Vaccination of pigs with a codon-pair bias de-optimized live attenuated influenza vaccine protects from homologous challenge. Vaccine. 36(8):1101-1107. https://doi.10.1016/j.vaccine.2018.01.027.
Walia, R.R., Anderson, T.K., Vincent, A.L. 2018. Regional patterns of genetic diversity in swine influenza A viruses in the United States from 2010 to 2016. Influenza and Other Respiratory Viruses.