Research Project: Intervention Strategies to Control Endemic and New Emerging and Re-Emerging Viral Diseases of Swine

Location: Virus and Prion Research

2022 Annual Report

Objectives
Objective 1. Elucidate the molecular mechanisms of PRRSV immunity. 1.A. Characterize virus-host interactions and determine innate and adaptive immune pathways that contribute to PRRSV disease susceptibility or immunity to inform the development of highly effective vaccines against very virulent strains. 1.B. Define mechanisms of immune evasion that contribute to PRRSV disease pathogenicity, and which can be targeted through recombinant vaccines to improve vaccine efficacy. Objective 2. Develop countermeasures to detect, prevent, and control endemic and emerging porcine coronaviruses. 2.A. Identify and characterize factors that determine coronavirus tissue and cellular tropism and adaptation to swine hosts. 2.A.2. Identify and characterize factors that determine coronavirus tissue and cellular tropism and adaptation to swine hosts. 2.B. Investigate and develop vaccine platforms that induce broadly cross-protective immune responses against PEDV, override PEDV vaccine interference from passively acquired immunity, and rapidly adapt to new and emerging porcine coronaviruses. 2.C. Determine genomic factors that drive coronavirus evolution and the mechanisms that lead to the emergence and spread of new porcine coronavirus strains. Objective 3. Predict and characterize the ecology and evolution of emerging viral diseases of swine. 3.A. Identify viral genes with mutations that are associated with SVA virulent and attenuated field strains and determine mechanisms of viral pathogenesis. 3.B. Conduct the molecular characterization of emerging SVA, including phylogenetic network analysis of viruses circulating in North America and Asia to predict the evolution of new SVA strains. 3.C. Develop SVA swine laboratory models to inform the development of vaccines. 3.D. Evaluate SVA new vaccine platforms and determine whether vaccines against SVA will cross-react with FMDV or interfere with FMDV serological surveillance. 3.E. Develop methods to rapidly detect and characterize the etiology of new and emerging viruses that may have an impact on swine health.

Approach
This research project will focus on swine diseases caused by viruses that are top concerns for United States pork producers: porcine reproductive and respiratory syndrome, porcine coronaviruses, and new and emerging diseases such as Seneca A virus. These pathogens will be examined in the laboratory as well as in swine disease models to investigate mechanisms of pathogenesis, transmission, immunity, evolution and methods of intervention. Animal experiments to be conducted involve one of three general designs: 1) disease pathogenesis and transmission studies, 2) vaccine efficacy studies, 3) sow/neonatal studies. Knowledge obtained will be applied to break the cycle of transmission of these swine pathogens through development of better vaccines or other novel intervention strategies. A major research approach will be the use of reverse engineering and infectious clones to identify virulence components of each virus under study through mutational studies. Development of vaccines that provide better cross-protective immunity than what is currently available with today’s vaccines will be approached through vaccine vector platform development, attenuated strains for vaccines and other novel technologies. A key approach in the study of disease pathogenesis is to better understand the host response to viral infection to various viruses. This research on comparative host transcriptomics will provide insights on viral pathogenesis and possible virulence factors that will enable rational design of more effective vaccines and target possible novel intervention strategies.

Progress Report
In support of Objective 1, using an established porcine reproductive and respiratory syndrome virus (PRRSV) cDNA infectious clone (pCMV-P129-GFP aka pKermit) gifted by Jay G. Calvert (Pfizer Animal Health, Kalamazoo, MI, USA) through an MTA with TSU conducted proof-of-concept studies in pigs using our modified live-virus (MLV) vaccine candidates created in a USDA-NIFA-AFRI funded research collaboration with Tennessee State University. Our vaccine candidates incorporated reverse-genetic modifications for replication-competent expression of several interferons (IFNs). This IFN cohort was selected based on our extensive functional characterization of the porcine IFN family. Compared with a commercial vaccine, we showed based on lower viral load and fever response that some vaccine candidates were effective in protecting pigs from challenge with a virulent field isolate. In support of Objective 3, conducted studies investigating the pathogenesis, protective immune response, evolution, and ecology of Senecavirus A (SVA). This virus emerged in 2015 and due to the similarity of SVA vesicular lesions to those caused by the foreign animal disease foot-and-mouth disease virus, it is of great concern for the swine industry. Results from these studies demonstrated: 1) the minimum infectious dose for SVA in neonatal pigs and market-weight pigs was approximately 10^3 median tissue culture infectious dose per milliliter; 2) maternal immunity generated by sows given two-doses of an inactivated whole-virus SVA vaccine provided protection to suckling piglets after SVA challenge; and 3) sequencing and phylogenetic analysis of SVA isolates from environmental swabs of sow slaughter facilities in 2020 demonstrated similarity of these isolates with other contemporary U.S. isolates, thus increasing the likelihood of cross-neutralizing antibodies. Information from these studies can be used to develop control and prevention measures to reduce the spread of SVA in the swine industry. In 2016 atypical porcine pestivirus (APPV) was demonstrated to be associated with congenital tremors (CT) in piglets. Collaboration with researchers at Iowa State University in Ames, Iowa, facilitated a longitudinal study following animals at a commercial farm and extended this work by transporting a subset of animals to the National Animal Disease Center (NADC) for further sampling. This research demonstrated that piglets born in APPV and CT positive litters tended over time to have a persistent viremia and a delay in detectable antibody response when compared to piglets born in APPV and CT negative litters. In addition, persistently infected animals bred at NADC were able to transmit virus to naïve contacts during gestation, but did not give birth to piglets with CT. In response to the COVID-19 pandemic and concerns about animals serving as a virus reservoir that could spill back into the human population, we have continued collaboration with scientists from Cornell University College of Veterinary Medicine in Ithaca, New York, to evaluate the pathogenesis of SARS-CoV-2 in white-tailed deer. Previous research performed at the NADC demonstrated white-tail deer fawns could replicate SARS-CoV-2 and transmit the virus to contact fawns; although, no clinical signs were observed. Follow-up work established that deer were only able to transmit virus to naïve contact animals during the first five days after experimental infection, adult deer had similar infection dynamics and shedding patterns compared to fawns, and there was limited genetic diversity in viruses isolated from nasal secretions of deer compared to the challenge virus. Therefore, white-tailed deer have the potential to serve as a viral reservoir for SARS-CoV-2 and further research will need to be performed to determine if deer will have an impact on the epidemiology of SARS-CoV-2 in humans.

Accomplishments
1. Determined the minimum infectious dose of Senecavirus A (SVA) in both neonates and market-weight pigs. Senecavirus A (SVA) is a causative agent for vesicular disease in swine. Vesicles are blister-like lesions that develop on the hoof of the pig. Despite efforts to control the spread of SVA in the swine industry, it continues to be detected triggering foreign animal disease investigations due to the similarity of SVA clinical signs to those caused by foot-and-mouth disease virus. To improve our understanding of the transmission of SVA, ARS researchers in Ames, Iowa, determined the minimum infectious dose (MID) for SVA in neonates and market-weight pigs was approximately 1000 tissue culture infectious viruses. This is important because pigs can shed SVA in vesicular fluids with each milliliter of fluid containing enough virus to potentially infect 1000 pigs. Veterinarians and pork producers may use MID information to design SVA disinfection and control measures to reduce the spread of SVA in the swine industry.

2. Demonstrated the efficacy of a whole-virus Senecavirus A (SVA) vaccine in generating maternal immunity that protected suckling piglets after SVA challenge. A whole-virus Senecavirus A (SVA) vaccine is effective in generating maternal immunity for protecting suckling piglets after SVA challenge. An efficacious and safe vaccine for SVA would provide the swine industry with a valuable tool to prevent the spread of SVA. There are no commercial vaccines currently available for SVA. Previous research performed at the NADC established that two doses of an inactivated SVA vaccine were able to prevent the development of vesicular lesions and significantly reduce viral shedding in nursery pigs experimentally challenged with SVA. Recently, ARS researchers in Ames, Iowa, demonstrated that two doses of an inactivated SVA vaccine also protected sows from the development of vesicular lesions after challenge. In addition, piglets suckling sows that had been vaccinated late in gestation did not replicate SVA after challenge, suggesting protective maternal immunity. Inactivated vaccines can be a safer alternative to modified live vaccines as they do not replicate in the host and thus cannot revert to virulence. Results from this study have shown that two doses of a whole-virus inactivated SVA vaccine could be used by the swine industry to prevent the incidence of vesicular lesions, improve animal welfare, and reduce the transmission of SVA in swine.

3. Optimized diagnostic assays for detection of antibodies against pseudorabies virus (PRV) in oral fluids. PRV has been eradicated from the U.S. domestic swine herd, but the virus can be found in some feral pig populations thus it is a constant threat to the commercial swine industry. Improving PRV detection and control efforts will involve rapid testing of large groups of swine, which can be done by testing oral fluid samples for the virus. Many enzyme-linked immunosorbent assays (ELISAs) utilize serum samples, which only provide information about individual animals. ARS researchers in Ames, Iowa, conducted a PRV challenge study to produce serum and oral fluids that were tested by a recombinant gE glycoprotein dual-matrix indirect ELISA. Results indicated the ELISA showed similar performance for both oral fluids and the more traditional serum sample; therefore, using oral fluids combined with the new test could be a useful tool for PRV surveillance and detection quickly for groups of animals. This will benefit veterinarians and pork producers as they improve rapid response plans in preparation for the possibility of the reintroduction of PRV into the commercial swine herd.

4. Created a novel modified-live vaccine (MLV) able to stimulate known antiviral interferons and examined for its ability to potentiate effective immunity and better protection. A new vaccine construct that stimulates known antiviral interferons potentiates effective immunity. Porcine reproductive and respiratory syndrome (PRRS) is the number one disease problem for US pigs. It is caused by the PRRS virus (PRRSV) for which there are commercially available vaccines. However, the vaccines only provide limited protection against the virus due to differences among PRRSV strains. Since vaccination remains the best available strategy for combatting PRRSV, we are conducting research into a new vaccine. ARS researchers in Ames, Iowa, in collaboration with researchers from Tennessee State University, evaluated a new modified-live vaccine (MLV) prototype able to stimulate known antiviral interferons (proteins made by the cell to combat viruses), and possibly increased cross-protection. The initial study demonstrated efficacy of the vaccine against one PRRSV strain setting the stage for subsequent studies to evaluate the extent of cross-protection induced by this vaccine. A PRRSV vaccine that induces better cross-protection would significantly reduce economic losses for pork producers and help provide a more stable supply of pork.

5. Investigated the host transcriptomic response in the thymus following porcine reproductive and respiratory syndrome virus (PRRSV) and influenza B virus (IBV) infections as well as their coinfection. Porcine Reproductive and Respiratory Syndrome Virus infection and coinfection with influenza B virus subvert a pig’s immune system in contrast to influenza B virus alone. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and influenza B virus (IBV) cause natural infections in pigs. PRRSV is the more common swine infection and as a primary infection, it can suppress the host immune system leaving pigs susceptible to secondary infections such as IBV or bacteria which can substantially increase the economic impact of the respiratory disease. Understanding how PRRSV induces immunosuppression is important because understanding this process may provide insight into development of new treatments or vaccines. One component of this immunosuppression is the ability of PRRSV to affect the thymus and impair its normal function in the immune response. ARS researchers in Ames, Iowa, investigated the host transcriptomic response (gene expression) in the thymus following PRRSV and IBV infections as well as their coinfection. In the thymus PRRSV infection downregulated genes involved in the immune response while IBV infection did not demonstrate the viruses have different mechanisms involved in causing respiratory disease in swine. Information from these basic studies can be applied to development of potential treatments or improved vaccines.

6. Foot-and-mouth disease (FMD) is one of the most significant constraints to international trade in animals and animal products described by the World Organization for Animal Health. To develop control strategies, including antiviral approaches and vaccines, it is essential to gain a better understanding of the virus-host cell interactions. SUMOylation, the covalent linkage of a small ubiquitin-like protein to a variety of substrate proteins, has emerged as an extensively studied posttranslational modification (PTM) that plays important roles in diverse biological processes. In this study, four lysine residues were found to jointly determine the SUMOylation of FMDV 3C protease. ARS researchers in Ames, Iowa, in collaboration with researchers at the Shandong Academy of Agricultural Sciences, demonstrated that SUMOylation attenuates FMDV 3C function, including the cleavage ability, inhibitory effect of interferon signaling pathway and protein stability, which in turn results in a decrease in FMDV replication. Our findings indicate that SUMOylation of FMDV 3C serves as a host cell defense against virus replication. In addition, ARS researchers in Ames, Iowa in collaboration with researchers at the Shandong Academy of Agricultural Sciences, demonstrated that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro relieves the inhibitory effect of sRNase L on production of ISGs and inhibits apoptosis through interacting with sRNase L. Further understanding of the cellular and molecular mechanisms driving this critical interaction should offer novel insights to design an effective strategy to control the dissemination of FMDV in animals.

Review Publications
Fleming, D.S., Miller, L.C., Li, J., Lager, K.M., Van Geelen, A., Sang, Y. 2022. Transcriptomic analysis of liver indicates novel vaccine to porcine reproductive and respiratory virus promotes homeostasis in T-Cell and inflammatory immune responses compared to commercial vaccine in pigs. Frontiers in Veterinary Science. 9. Article 791034. https://doi.org/10.3389/fvets.2022.791034.
Sang, Y., Miller, L.C., Nelli, R.K., Giménez-Lirola, L.G. 2021. Harness organoid models for virological studies in animals: A cross-species perspective. Frontiers in Microbiology. 12. Article 725074. https://doi.org/10.3389/fmicb.2021.725074.
Khwatenge, C.N., Pate, M., Miller, L.C., Sang, Y. 2021. Immunometabolic dysregulation at the intersection of obesity and COVID-19. Frontiers in Immunology. 12. Article 732913. https://doi.org/10.3389/fimmu.2021.732913.
Sui, C., Jiang, D., Wu, X., Liu, S., Li, F., Pan, L., Cong, X., Li, J., Yoo, D., Rock, D.L., Miller, L.C., Lee, C., Du, Y., Qi, J. 2021. Inhibition of antiviral innate immunity by Foot-and-Mouth Disease Virus Lpro through interaction with N-terminal domain of swine RNase L. Journal of Virology. 95(15). https://doi.org/10.1128/JVI.00361-21.
Hau, S.J., Devries, A.C., Brockmeier, S. 2022. Bacterin vaccination provides insufficient protection against Streptococcus equi subspecies zooepidemicus infection in pigs. Frontiers in Veterinary Science. 9. Article 827082. https://doi.org/10.3389/fvets.2022.827082.
Devries, A.C., Lager, K.M. 2022. Efficacy of an inactivated Senecavirus A vaccine in weaned pigs and mature sows. Vaccine. 40(12):1747-1754. https://doi.org/10.1016/j.vaccine.2022.02.018.
Martins, M., Boggiatto, P.M., Buckley, A., Cassmann, E.D., Falkenberg, S.M., Caserta, L.C., Fernandes, M.H., Kanipe, C.R., Lager, K.M., Palmer, M.V., Diel, D.G. 2022. From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in highly susceptible white-tailed deer. PLoS Pathogens. 18(3). Article e1010197. https://doi.org/10.1371/journal.ppat.1010197.
Hoffman, K., Humphrey, N., Korslund, J., Anderson, T.K., Faaberg, K., Lager, K.M., Devries, A.C. 2022. Characterization of Senecavirus A isolates collected from the environment of U.S. sow slaughter plants. Frontiers in Veterinary Science. 9. Article 923878. https://doi.org/10.3389/fvets.2022.923878.
Devries, A.C., Lager, K.M. 2022. Infectious dose of Senecavirus A in market weight and neonatal pigs. PLoS ONE. 17(4). Article e0267145. https://doi.org/10.1371/journal.pone.0267145.
Devries, A.C., Lager, K.M. 2022. Senecavirus A: Frequently asked questions. Swine Health and Production. 30(3):149-159. https://doi.org/10.54846/jshap/1270.
Cheng, T., Magtoto, R., Henao-Diaz, A., Poonsuk, K., Devries, A.C., Van Geelen, A., Lager, K.M., Zimmerman, J., Gimenez-Lirola, L. 2021. Detection of pseudorabies virus antibody in swine serum oral fluid specimens using a recombinant gE glycoprotein dual matrix indirect ELISA. Journal of Veterinary Diagnostic Investigation. 33(6). p. 1106-1114. https://doi.org/10.1177/10406387211040755.