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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

2020 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 identify genes associated with quantity and duration of virus shedding and test up- or down-regulation of genes associated with virus replication in vitro with viruses of different genotypes, messenger RNA was extracted from porcine alveolar macrophages and lung tissue from pigs infected with influenza A virus (IAV) from a previously completed study. Gene expression from infected pigs were compared to uninfected pigs using transcriptomic sequencing techniques. Differences in gene expression patterns were observed; however, progress was slowed due to project plan vacancy. In support of Objective 1, Subobjective 1.3, to identify genetic signatures associated with inter-species adaptation in swine, four conserved hemagglutinin (HA) amino acid mutations were identified in swine 2010.1 field H3N2 strains that were distinct from human seasonal precursor H3. The role of these four HA amino acids in the adaptation of human H3 to swine was evaluated in reverse engineered viruses containing HA genes with site-directed mutagenesis, alone or in combination. An H3 gene from a human seasonal IAV was mutated to contain the four swine-like amino acids and an H3 gene from a swine IAV was mutated to contain the four human-like amino acids. Mutant viruses were tested in vitro and in vivo. In support of Objective 2, Subobjective 2.1a, to conduct whole genome analyses and develop or extend existing methods to identify linked evolution within and between gene segments, comprehensive phylogenetic 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 HA and NA phylogenetic clade combinations. Constellations were partitioned by state and clustered by distance metrics to identify temporal and spatial patterns. An evolutionary distance-based method was developed to identify within-lineage reassortment events among strains. An algorithm that merged the evolutionary history of individual genes into a larger phylogenetic network describing the evolution of viruses was developed. Representative viruses detected with sequence analysis pipelines were selected for antigenic analysis using swine and ferret antisera. An in vivo pig study was completed to evaluate pathogenesis and transmission of 3 IAV from swine with differing whole genome gene constellations. In support of Objective 2, Subobjective 2.1b, to develop automated tools that perform routine sequence analysis of IAV-S surveillance sequence data to monitor for genetic and potential antigenic evolution, comprehensive phylogenetic analyses were conducted on publicly available swine IAV genes. An automated pipeline was created to assign evolutionary lineage and genetic clade to query gene segments (octoFLU) and a graphical web interface to visualize phylogenetic categorization is in development (octoFLU-SHOW) on a public-facing website ( The website is hosted on Amazon Web Services as part of the ARS Big Data Initiative, SCINet. To integrate genetic and antigenic data, a novel network NoSQL database is in development to support octoFLU-SHOW. In support of Objective 2, Subobjective 2.2, to monitor contemporary viruses for antigenic evolution and test amino acid substitutions in H1 for antigenic evolution, wild type viruses were selected from the USDA influenza A virus swine surveillance system for testing. Contemporary IAV from U.S. swine were tested by hemagglutination inhibition (HI) assays and by neuraminidase inhibition assays. An in vivo pig study to test the antibody response to a sequence of vaccination with heterologous H1 viruses was conducted. To identify critical amino acid positions that influence the IAV phenotype, machine learning models were parameterized by HI data. Models demonstrated that HA gene sequence identity and mutations at ten amino acid positions could predict the antigenic distance between IAV strains. An ensemble machine learning model was then used to predict the phenotype of four uncharacterized viruses, which were then evaluated by HI assays. In support of Objective 3, Subobjective 3.2, to test LAIV engineered to carry immunomodulatory genes to improve heterologous protection, an in vivo pig vaccination study was conducted. In collaboration with University of Georgia, a bivalent reverse engineered LAIV with H1N1 and H3N2 strains expressing a synthetic swine IgA-inducing protein (IGIP) was generated and tested in pigs. The LAIV with IGIP were compared to LAIV without IGIP and to nonvaccinated pigs. A vaccine study to compare cell mediated immunity between different vaccine platforms was also completed.

1. Development of octoFLU: An automated classification for evolutionary origin of influenza A virus gene sequences. Influenza A virus (IAV) in swine is a respiratory disease that can be divided into different subtypes based on the composition of genes contained within the virus. Although only H1N1, H1N2, and H3N2 subtypes of IAV are found in swine around the world, much diversity can be detected in the genes coding for the major surface proteins, hemagglutinin (H) and neuraminidase (N), and in the other 6 internal gene segments. This diversity is the result of frequent bidirectional transmission between swine and humans, and the occasional transmission of an avian virus into swine followed by periods of virus evolution. Using a curated dataset of whole genome swine IAV sequences, ARS scientists in Ames, Iowa, developed a publicly available and adaptable software pipeline that rapidly assigns evolutionary origin to swine IAV gene sequences. This tool will aid agricultural production and diagnostic capabilities through the identification of important changes in genetic diversity, and allow for the identification of novel viruses.

2. A serosurvey for bovine influenza D virus exposure from cattle across the United States, 2014-2015. An emerging type of influenza, Influenza D virus (IDV), has been detected in cattle from several countries. In the USA, regional and state level IDV prevalence has been previously reported, but little information exists to evaluate national prevalence. Thus, a study was performed to detect antibodies against IDV with approximately 2,000 bovine serum samples collected in 2014 and 2015 from across the USA. ARS scientists in Ames, Iowa, demonstrated that IDV had circulated in the sampled cattle population, causing a high overall percentage of positive (77.5%) countrywide, from 41 states of the 42 states sampled. The ubiquitous distribution of IDV in cattle from USA highlights a need for greater understanding of IDV pathogenesis, epidemiology and the impact on animal health for cattle ranchers and veterinary clinicians and diagnosticians.

3. Swine influenza A viruses have a tangled relationship with humans. Influenza A viruses (IAV) are the causative agents of one of the most important viral respiratory diseases in pigs and humans. Human and swine IAV are prone to interspecies transmission, leading to regular incursions from human to pig and vice versa. Transmission of distinct human seasonal IAV to pigs, followed by sustained within-host transmission and rapid adaptation and evolution represent a considerable challenge for pig health and production. Consequently, although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine around the world, extensive diversity was found in the hemagglutinin (HA) and neuraminidase (NA) genes, as well as the remaining 6 genes. ARS scientists in Ames, Iowa, demonstrated that the complicated global epidemiology of IAV in swine and the implications for public health and influenza pandemic planning are inextricably entangled. These results aid in pandemic planning for the human population and in developing control strategies in swine populations, as well as highlight the need for continued surveillance, research, and collaboration in both species.

4. Influenza A virus field surveillance and sequencing at a swine-human interface. Influenza A virus (IAV) is an important pathogen of swine and humans. Swine and humans can share some strains of IAV, and swine IAV can lead to sporadic human infection, called "variant" IAV in humans. These variant transmission events are often associated with animal agricultural exhibits and swine exposure. ARS scientists in Ames, Iowa, collaborated with public and animal health researchers to develop and deploy a rapid and mobile sequencing method for identifying and characterizing IAV detected in pigs at a public swine exhibit. Using this technology on-site at the exhibit, 13 virus genomes from infected swine were sequenced and characterized within 18 hours, dramatically reducing time to diagnosis and allowing time for intervention and mitigation of further transmission to other swine and/or humans in contact with the infected pigs on-site. This technology will benefit animal health officials at livestock exhibits and shows. Additionally, this information will inform vaccine strain selection for swine and for humans as part of pandemic preparedness.

5. Detection of live attenuated influenza vaccine virus and evidence of reassortment in the U.S. swine population. Influenza A virus (IAV) is an important respiratory disease of swine that can cause significant economic losses for producers. The genome of IAV contains 8 gene segments that can be mixed when an individual is infected with more than one strain of IAV. This process, called reassortment, results in new gene combinations in progeny viruses that may escape protection from prior vaccination. Inactivated and live attenuated influenza virus vaccines (LAIV) are available in the U.S. for use in swine. In this study, the genetic makeup of IAV that were detected in swine diagnostic submissions in the U.S. during 2018 were examined. ARS scientists in Ames, Iowa, identified gene combinations in viruses that were generated by reassortment events between circulating swine IAV and the strains included in the LAIV. These reassorted genotypes represent unique viruses compared to other recently circulating IAV and may not be recognized by immunity against contemporary circulating strains. The reassortment between the LAIV strains and contemporary IAV increased the genetic diversity among swine IAV, with potential impact to the swine industry if these novel strains spread to non-LAIV vaccinated herds. These findings are important for informed vaccine selection for swine herds and contribute to the understanding of swine influenza evolution for animal and public health.

6. A swine-origin H3N2 closely related to human H3N2v demonstrated transmission from swine to ferrets. The transmission of influenza A viruses (IAV) from swine to humans occurs sporadically and is often associated with agricultural fairs in the USA. Swine origin IAV that are detected in humans are called "variant" to differentiate from human seasonal IAV. During the 2016-2017 influenza season, 61 H3N2 variant (H3N2v) cases were reported. ARS scientists in Ames, Iowa, compared the genomes of human H3N2v viruses and swine H3N2 viruses collected at the same state fair in Ohio. Ferrets were directly infected with the H3N2 virus from the 2017 fair outbreak, showing efficient infectivity. Additionally, pigs were infected with the virus, and placed in an enclosure in close proximity to caged ferrets, the gold-standard model of human IAV infection and transmission. The swine H3N2 replicated in pigs and the ferrets that were exposed to the infectious respiratory aerosols of the pigs, showing potential transmission from pigs to susceptible ferrets as a surrogate model for humans. A temporal whole genome sequence dataset of IAV collected from infected pigs and caged ferrets was published at the National Agricultural Library. These results are the first to show a transmission model from swine to ferrets without modification to the virus and highlight the need to reduce swine IAV at animal exhibits. This study also demonstrates the importance of continued surveillance, research, and collaboration on swine and human IAV.

7. Epidemiologic and phylogenetic analyses of influenza A viruses (IAV) provide rational criteria for vaccine strain selection and may identify viruses with pandemic potential. All circulating swine IAV have gene segments derived from human seasonal IAV, and these swine IAV have the potential to be introduced back into the human population if they are substantially different from current human seasonal strains. ARS scientists in Ames, Iowa, quantified the global genetic diversity of swine IAV circulating from 2016 to present, and the genetic diversity of swine IAV in the USA over the past 6 months. The circulating swine IAV diversity was compared to human IAV vaccines and current candidate vaccine viruses (CVV) that are used in pandemic preparedness. Representative strains were antigenically characterized to determine whether human vaccine strains or CVV strains would provide protection against these swine IAV. Only 7 of the 29 distinct circulating genetic clades were covered by CVV strains; and the degree to which those vaccines provide protection is dubious given observed genetic and antigenic differences. This work demonstrated that controlling IAV in swine populations is a critical process in pandemic preparedness, and objectively identified vaccine strain candidates for the benefit of public health pandemic preparedness.

8. Coordinated evolution between influenza A virus surface proteins. Influenza A virus (IAV) is an important respiratory pathogen in swine with significant economic losses due to decreased rate of weight gain, increased antibiotic and vaccine costs, and increased mortality. Vaccine strategies to control infection have focused on the hemagglutinin (HA) protein, but efficacy is challenged by continual genetic change that interferes with vaccine-induced immunity or prior infection. 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. ARS scientists in Ames, Iowa, genetically characterized N2 NA genes circulating between 2010 and 2018 in US swine. These data revealed increases in the diversity of the N2 gene, with continual circulation of multiple genetic groups. NA genes were shown to be preferentially paired with different HA genes and demonstrated how NA-HA pairing results in rapid evolutionary changes in both proteins. These data also showed how the interstate movement of pigs and their viruses resulted in novel NA-HA pairings, and this movement spurred rapid changes in genetic diversity, along with the introduction of novel genes into new regions. This study developed new classifications for N2 genes, demonstrates how NA-HA evolution are paired, and provides critical information for manufacturers and producers on how to objectively develop better vaccines with field-relevant components.

Review Publications
Chang, J., Anderson, T.K., Zeller, M.A., Gauger, P.C., Vincent, A.L. 2019. OctoFlu: Automated classification of influenza A virus gene sequences detected in U.S. swine to evolutionary origin. Microbiology Resource Announcements. 8(32):e00673-19.
Rambo-Martin, B.L., Keller, M.W., Wilson, M.M., Nolting, J.M., Anderson, T.K., Vincent, A.L., Bagal, U., Jang, Y., Neuhaus, E.B., Davis, C.T., Bowman, A.A., Wentworth, D.E., Barnes, J.R. 2020. Influenza A virus field surveillance at a swine-human interface. mSphere. 5(1):e00822-19.
Anderson, T.K., Chang, J., Arendsee, Z., Vekatesh, D., Souza, C.K., Kimble, B., Lewis, N., Davis, C., Vincent, A.L. 2020. Swine influenza A viruses and the tangled relationship with humans. In:Neumann, G. , Kawaoka, Y., editors. Influenza: The Cutting Edge. Cold Spring Harbor Perspectives in Medicine. Cold Spring Harbor Laboratory Press.10(7):p.a038737.
Sharma, A., Zeller, M.A., Li, G., Harmon, K., Zhang, J., Hoang, H., Anderson, T.K., Vincent, A.L., Gauger, P.C. 2020. Detection of live attenuated influenza vaccine virus and evidence of reassortment in U.S. swine population. Journal of Veterinary Diagnostic Investigation. 32(2):301-311.
Silveira, S., Falkenberg, S.M., Kaplan, B.S., Crossley, B., Ridpath, J.F., Bauermann, F.B., Fossler, C.P., Dargatz, D.A., Dassanayake, R.P., Vincent, A.L., Canal, C.W., Neill, J.D. 2019. Serosurvey for influenza D virus exposure in cattle, United States, 2014-2015. Emerging Infectious Diseases. 25(11).
Zeller, M.A., Chang, J., Vincent, A.L., Gauger, P.C., Anderson, T.K. 2020. Coordinated evolution between N2 neuraminidase and H1 and H3 hemagglutinin genes increased influenza A virus genetic diversity in swine. Molecular Biology and Evolution.
Kaplan, B.S., Kimble, J.B., Chang, J., Anderson, T.K., Gauger, P., Janas-Martindale, A., Killian, M., Bowman, A.S., Vincent, A.L. 2020. Aerosol transmission from infected swine to ferrets of a swine H3N2 virus collected from an agricultural fair and associated with human variant infections. Journal of Virology.