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

Location: Virus and Prion Research

2019 Annual Report

Objectives
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.

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 test wildtype and/or engineered viruses in screening assays, four conserved HA amino acid mutations were identified in 2010.1 swine field strains that were distinct from human seasonal H3. Using site-directed mutagenesis in various in vitro assays, these four HA amino acids were evaluated in the adaptation of human H3 to swine. Assays included hemagglutinin receptor binding avidity, surfactant protein D neutralization, virion stability in a thermal stability HA assay, and replication in primary porcine respiratory cell cultures. In support of Objective 1, Subobjective 1.3, to test reverse engineered or naturally occurring viruses in vivo, reverse engineered H3N1 viruses with mutations in the HA were tested to identify molecular determinants for adaptation of human viruses to swine. Four mutations in the HA in the background of swine adapted H3 and in the precursor human seasonal H3 were identified to be important for swine adaptation of human seasonal H3 using in vitro methods. A pig pathogenesis and transmission study was conducted. 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 IAV 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 gene reassortment events among strains within the same clade. In support of Objective 2, Subobjective 2.1b, to automate perpetually updated phylogenies for swine IAV H1 and H3 with annotated molecular signatures, an automated annotation tool was developed to assign H3 lineage categories to sequence data and an adaptable bioinformatic pipeline was developed that rapidly assigns genetic origin and host to query swine influenza A virus genome sequences. Co-circulating swine H3 lineages were identified by the H3 tool and criteria were also included to classify human, avian, canine, and equine H3-HA sequences. In support of Objective 2, Subobjective 2.2, to monitor contemporary viruses for antigenic evolution, wild type viruses were selected from the USDA influenza A virus swine surveillance system for testing. Contemporary 2010.1 and 2010.2 H3N2 viruses and gamma (1A.3.3.3) and delta (1B.2.1 and 1B.2.2) H1 viruses were tested by hemagglutination inhibition assays. Antigenic evolution was also evaluated in N2 by neuraminidase inhibition assays. Antigenic distance was quantified by antigenic cartography. In support of Objective 3, Subobjective 3.1, to evaluate the role of viral proteins and/or route of administration in the mechanism of Vaccine-Associated Enhanced Respiratory Disease (VAERD), an in vivo study was conducted to evaluate vectored HA and NA vaccines. Pigs were vaccinated with replicon particle vaccines expressing matched or mismatched HA in combination with matched NA and challenged with H1N1. The vectored vaccines were also compared to whole inactivated vaccines.

Accomplishments
1. Vaccine adjuvant impacted vaccine-associated enhanced respiratory disease in pigs. Influenza A viruses (IAV) cause an important respiratory disease in pigs and a significant economic burden for the swine industry. Vaccines are commonly used in swine to protect against IAV but are often ineffective due to the diversity in strains that circulate in U.S. pig populations. Whole inactivated virus (WIV) with adjuvants are the traditional vaccine for use in swine. One type of adjuvant, an oil-in-water (OW) emulsion, is commonly used in the swine industry, but WIV-OW has been associated with vaccine enhanced respiratory disease (VAERD). ARS scientists in Ames, Iowa, showed the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar IAV, but only OW was associated with VAERD. These results highlight the impact adjuvant can have on vaccine effectiveness and provide important information for improving vaccines against IAV for use in swine.

2. H3N2 variant influenza A virus outbreaks of swine origin were detected in three fairs in 2017. Influenza A virus (IAV) is a significant health burden to human and swine populations, and IAV are sporadically shared between the two hosts. When humans are infected with a swine IAV, it is termed “variant” to differentiate it from typical human seasonal strains. In September 2017, a joint investigation of outbreaks of influenza-like illness (ILI) in exhibited swine and in exhibitors and attendees at 3 Maryland county fairs was conducted. Forty people were subsequently confirmed influenza A (H3N2) variant virus positive. Samples were collected from pigs at 2 of the 3 fairs and tested positive for H3N2. 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. This outbreak investigation emphasizes the collaborative, One Health approach needed to investigate and respond to variant influenza virus outbreaks, including the application of both swine and human infection control measures.

3. Comparison of H3N2 influenza A virus vaccines in pigs. Due to the rapid evolution of the influenza A virus, vaccines require continuous strain updates. The platform used to deliver the vaccine can have an impact on the breadth of protection. Various vaccine platforms are available to prevent influenza A virus infection in swine, and ARS scientists in Ames, Iowa, experimentally tested two: adjuvanted-whole inactivated virus and live attenuated virus. When challenged with an antigenically distinct virus, adjuvanted-whole inactivated virus provided partial protection while live attenuated virus provided effective protection. Enhancing vaccine efficacy to control influenza infections in swine will help reduce the impact it has on swine production and reduce the risk of swine-to-human transmission.

4. Development of a web-based platform to monitor influenza A virus patterns in swine. Influenza A Virus (IAV) causes respiratory disease in swine, impacting health and production. Cases of respiratory disease in swine are submitted to state veterinary diagnostic laboratories (VDL) for testing and sequencing to inform vaccine selection. Iowa State University (ISU) VDL possessed a database of influenza diagnostic test results and sequences accumulated since 2003, but these data were not publicly available. Therefore, in a collaboration between ISU, VDL, and ARS scientists in Ames, Iowa, FLUture was created, a web portal to summarize and graphically visualize IAV case and genetic sequence information derived from swine farms across the United States of America. This rapid access information about IAV in swine provides rational criteria to inform vaccine design and current control efforts.

5. Novel human-like H3N2 influenza A viruses detected in swine. Influenza A virus (IAV) is an RNA virus that can be shared between humans and pigs and is a cause of respiratory disease in both. Spillover of human seasonal IAV into swine occurs sporadically, resulting in the emergence of novel strains and increased genetic diversity of IAV maintained in swine. ARS scientists in Ames, Iowa, with collaborators at Iowa State University described the detection of two novel swine IAV in 2017 containing human seasonal influenza gene segments. Two novel human-like H3N2 influenza A viruses were isolated and whole genome sequences were reported. This report provides evidence of a recent human-to-swine transmission of a human seasonal H3N2 and underscores the importance of preventing human IAV transmission to swine in production facilities.

6. A single amino acid position in the H3 hemagglutinin protein of swine influenza A virus has high flexibility and impact. Influenza A virus (IAV) is an endemic and economically important pathogen in pigs with the potential to infect humans. The hemagglutinin (HA) protein on the surface of IAV binds to host cells. Amino acid substitutions in the HA allows IAV to escape population immunity through antigenic drift. The HA protein is the primary target of protective immune responses and the major component in swine IAV vaccines. ARS scientists in Ames, Iowa, confirmed that a small set of positions in the HA protein are largely responsible for driving antigenic drift in swine-origin H3 IAV, namely at amino acid position 145. Functional constraints to substitutions at 145 and their impact on receptor binding and antigenic properties were tested. Many substitutions were tolerated and stably maintained without major impact on virus replication. All substitutions retained receptor binding specificity, but frequently led to decreased receptor binding or modulated binding profiles. Antigenic characterization identified specific substitutions that altered antibody recognition. This work provides a better understanding of the impact of substitutions in the HA on the interplay between receptor binding and antigenic drift and can help inform vaccine strain selection.

7. Evolution of H3N2 in swine in the United States between 2012 and 2015. Influenza A virus (IAV) is an important respiratory pathogen in swine. There is extensive diversity in IAV that can interfere with immunity from vaccination or prior infection. Genetic characterization of contemporary hemagglutinin gene sequences from H3 IAV in swine revealed increased diversity at specific sites within the gene that are associated with antibody recognition. These specific sites in the H3 sequences were used to predict antibody recognition, viruses were characterized to test the predictions, and antigenic properties were defined based on these results. ARS scientists in Ames, Iowa, found many antigenically distinct viruses circulating in the United States swine population, but that sequence-based predictions of antigenic phenotype are possible. Understanding antigenic diversity in swine IAV has important implications for effective vaccine design, and the findings from this study are critical to help inform vaccine manufacturers and swine producers on how to develop and implement vaccines to reduce circulation of this important pathogen in swine.

8. Alphavirus-vectored hemagglutinin (HA) subunit vaccine provided partial protection against heterologous challenge in pigs. Development of broadly protective vaccine against influenza A virus (IAV) in swine is challenging because the genetic and antigenic diversity is large. Effective use of vaccines can help reduce the burden of disease, and there are multiple vaccine platforms commercially available. In an experimental vaccine challenge study, ARS scientists in Ames, Iowa, compared monovalent and bivalent HA subunit vaccines. We found that monovalent HA vaccines provided efficient protection against antigenically distinct HA viruses and that bivalent vaccines elicited an antibody response to both antigens and provided efficient protection. Examining the efficacy of available vaccine platforms helps inform swine producers on how to best implement vaccine use to reduce IAV infections in swine.

9. Human-origin influenza A (H3N2) viruses in swine in Southeast Mexico. Influenza A viruses (IAVs) that circulate in swine have the potential to infect humans and may cause pandemics, as in 2009. The 2009 H1N1 pandemic likely originated in swine in Mexico, yet the level of surveillance for IAV in swine in Mexico is relatively low. Available data suggested that some IAV in swine in Mexico may be similar to those in the United States, but some may be unique to Mexico. Producers attempt to control IAVs in their herds through ongoing monitoring and vaccination, but vaccines should be matched to the circulating strains. Along with collaborators, ARS scientists in Ames, Iowa, characterized IAVs collected from swine from multiple regions in Mexico and identified extensive viral genetic and antigenic diversity, including strains that are highly divergent from those circulating in United States herds. As such, vaccine intervention for IAV in swine herds in Mexico must be tailored specifically to the country’s strains. This information is important for effective control of IAV in swine.

10. An avian influenza virus A (H7N9) that emerged in the United States in 2017 did not efficiently infect swine. Influenza A virus (IAV) is an important pathogen in humans, pigs and birds among other species, and IAV strains that are adapted to one species can sporadically infect a different species and cause outbreaks. There is a potential risk of avian viruses from wild birds spilling over into swine due to overlap between pig production systems and major bird migration flyways, as well as pig and poultry production regions. In Spring of 2017 a novel avian-origin H7N9 virus was detected in poultry in Tennessee, Georgia, Alabama and Kentucky and caused significant economic loss. In an experimental challenge study, ARS scientists in Ames, Iowa, were unable to detect virus in the lungs or in the nasal cavity, and there was no evidence of pig-to-pig transmission. These findings suggest that there is a low probability the avian-origin H7N9 could be sustained in the pig population without substantial evolutionary changes to the genetics of the virus indicating this lineage of avian virus poses negligible risk to U.S. swine.

11. 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 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., Rajao, D.S., Santos, J., Kaplan, B.S., Nicholson, T.L., Brockmeier, S.L., Gauger, P.C., Perez, D.R., Vincent, A.L. 2018. Comparison of adjuvanted-whole inactivated virus and live-attenuated virus vaccines against challenge with contemporary, antigenically distinct H3N2 influenza A viruses. Journal of Virology. 92(22):e01328-18. https://doi.org/10.1128/JVI.01323-18.
Souza, C.K., Rajão, D.S., Sandbulte, M.R., Lopes, S., Lewis, N.S., Loving, C.L., Gauger, P.C., Vincent, A.L. 2018. The type of adjuvant in whole inactivated influenza A virus vaccines impacts vaccine-associated enhanced respiratory disease. Vaccine. 36(41):6103-6110. https://doi.org/10.1016/j.vaccine.2018.08.072.
Duwell, M., Blythe, D., Radebaugh, M.W., Kough, E.M., Bachaus, B., Crum, D.A., Perkins Jr., K.A., Blanton, L., Davis, T., Jang, Y., Vincent, A.L., Chang, J., Abney, D.E., Gudmundson, L., Brewster, M.G., Polsky, L., Rose, D., Feldman, K.A. 2018. Influenza A (H3N2) variant virus outbreak at three fairs - Maryland, 2017. Morbidity and Mortality Weekly Reports. 67(42):1169-1173. https://doi.org/10.15585/mmwr.mm6742a1.
Zeller, M.A., Anderson, T.K., Walia, R.W., Vincent, A.L., Gauger, P.C. 2018. ISU FLUture: a veterinary diagnostic laboratory web-based platform to monitor the temporal genetic patterns of influenza A virus in swine. BMC Bioinformatics. 19:397. https://doi.org/10.1186/s12859-018-2408-7.
Santos, J., Abente, E.J., Obadan, A.O., Thompson, A.J., Ferreri, L., Geiger, G., Gonzalez-Reiche, A.S., Lewis, N.S., Burke, D., Rajão, D.S., Paulson, J.C., Vincent, A.L., Perez, D.R. 2018. Plasticity of amino acid residue 145 near the receptor binding site of H3 swine influenza A viruses and its impact on receptor binding and antibody recognition. Journal of Virology. 93(2):e01413-18. https://doi.org/10.1128/JVI.01413-18.
Bolton, M.J., Abente, E.J., Venkatesh, D., Stratton, J.A., Zeller, M., Anderson, T.K., Lewis, N.S., Vincent, A.L. 2018. Antigenic evolution of H3N2 influenza A viruses in swine in the United States from 2012–2016. Influenza and Other Respiratory Viruses. 13:83-90. https://doi.org/10.1111/irv.12610.
Rajao, D.S., Vincent, A.L., Perez, D.R. 2019. Adaptation of human influenza viruses to swine. Frontiers in Veterinary Science. 5:347. https://doi.org/10.3389/fvets.2018.00347.
Abente, E.J., Rajao, D.S., Gauger, P.C., Vincent, A.L. 2019. Alphavirus-vectored hemagglutinin subunit vaccine provides partial protection against heterologous challenge in pigs. Vaccine. 37(11):1533-1539. https://doi.org/10.1016/j.vaccine.2018.12.071.
Nelson, M.I., Souza, C., Trovão, N.S., Diaz, A., Mena, I., Rovira, A., Vincent, A.L., Torremorell, M., Marthaler, D., Culhane, M.R. 2019. Human-origin influenza A (H3N2) reassortant viruses in swine, southeast Mexico. Emerging Infectious Diseases. 25(4):691-700. https://doi.org/10.3201/eid2504.180779.
Powell, J.D., Abente, E.J., Torchetti, M.K., Killian, M.L., Vincent, A.L. 2019. An avian influenza virus A (H7N9) reassortant that recently emerged in the United States with low pathogenic phenotype does not efficiently infect swine. Influenza and Other Respiratory Viruses. 13:288-291. https//doi.org/10.1111/irv.12631.
Zeller, M.A., Li, G., Harmon, K.M., Zhang, J., Vincent, A.L., Anderson, T.K., Gauger, P.C. 2018. Complete genome sequences of two novel human-like H3N2 influenza A viruses A/swine/Oklahoma/65980/2017(H3N2) and A/swine/Oklahoma/65260/2017(H3N2) detected in swine in the United States. Microbiology Resource Announcements. 7(20):e01203-18. https://doi.org/10.1128/MRA.01203-18.