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

Location: Virus and Prion Research

2021 Annual Report

Objectives
1. Identify pathogenic mechanisms of swine Nidovirales, including identifying the pathogenic mechanisms of Porcine Respiratory and Reproductive Syndrome Virus (PRRSV), and the pathogenic mechanisms of Porcine Epidemic Diarrhea Virus (PEDV). 2. Discover and assess vaccines that can reduce or prevent economic losses from swine viral diseases, including identifying mechanisms to modulate innate and adaptive immune responses to swine viral pathogens and investigating technologies to override vaccine interference from passively acquired immunity. 3. Determine evolutionary antigenic and pathogenic properties of economically significant swine viral pathogen, including identifying and monitoring genetic and antigenic evolution in Nidovirales and emerging viral pathogens. 4. Identify mechanisms of pathogenesis, transmission, and immunity for emerging viral diseases of swine, starting with evaluating the onset and duration of Seneca A virus immunity in swine.

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
This is the final report for this project 5030-32000-118-00D terminating October 18, 2021. Over the course of the project inter-related experiments were conducted that would address more than one objective at a time. In support of Objective 1, a series of studies were completed in collaboration with researchers at Loyola University studying how porcine coronaviruses cause disease in swine. This research included development of infectious clones of porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus virus (PDCoV), two porcine coronaviruses that are capable of infecting and causing severe disease in young swine. Infectious virus could be rescued from the clones for experimental studies. Mutations were introduced into both rescued viruses that would reduce the capacity of each virus to cause disease in baby pigs, the most susceptible age of swine. Subsequent studies investigated the potential of these mutated viruses to serve as modified-live-virus vaccines resulting in one patent application being submitted. Additional studies are underway investigating possible changes/mutations to the current attenuated viruses for possible use as a live virus vaccine in sows. Porcine reproductive and respiratory syndrome (PRRS) is the number one problem for U.S. pork producers. This disease is caused by PRRS virus which was discovered about 30 years ago. Although much is known about the virus and disease, it is still difficult to control PRRS indicating that new thoughts and approaches are needed. In support of Objective 2, we conducted a series of studies investigating different strategies to develop novel vaccine concepts that could improve upon currently available PRRSV vaccines. This research also included investigating the molecular response of the pig to PRRSV infection, and comparison of this response to other swine virus infections with the goal of using this new understanding to improve vaccines. One approach produced a PRRSV chimera where the first three-fourths of the genome consisted of vaccine sequence including an immunogenic tag and the remaining sequence was that of a recent field strain (NADC34). The chimera was passed in the presence of the parental vaccine at varying ratios to derive viral recombinants. The viral chimera was also inoculated into pigs and compared to the native field strain, parental vaccine, and the vaccine with an immunogenic tag. The chimeric virus is a candidate vaccine with the ability to differentiate vaccinated from infected animals. In addition, a new NADC 34 clone was made in a different vector that could more easily produce infectious virus when transfected directly into mammalian cells or by producing RNA transcripts that are then transfected. This infectious clone will be used to prepare new vaccine formulations to evaluate. Another approach examined the growth of PRRSV strains in porcine alveolar macrophages that had double or quadruple amino acid changes in the replicase protein known as nonstructural protein 2. The various combinations of amino acid mutations had previously been found to control the levels of inhibition of interferon stimulating gene 15 and/or ubiquination, a cellular process that can help protect the cell from a virus infection. Porcine alveolar macrophages were not sensitive enough to define a difference in a set of interferon responsive genes after infection with the parent viral strains, Ingelvac PRRSV modified live vaccine and highly pathogenic JXwn06. Swine were found to replicate the altered viruses as well as the parent strains and these altered viruses retained the mutations over the course of the study. A series of studies were completed to analyze the molecular response to common swine viral pathogens with the goal of analyzing regulation of specific genes in response to each virus infection. To acquire a better understanding of the molecular host response related to PRRS disease, gene expression changes that occur in tracheobronchial lymph nodes (TBLN) of pigs infected with either PRRSV, porcine circovirus type 2 (PCV2), or swine influenza A virus (IAV-S) infections were compared by Digital Gene Expression Tag Profiling (DGETP). Experimental infections with these viruses (PRRSV, IAV-S and PCV-2) induced the expected clinical disease and the DGETP studies revealed expected and unexpected changes in gene expression following infection that helped define virus-specific responses. Additional experiments investigated the effect of PRRSV on monocytic immune cells, the cells in swine that are susceptible to PRRSV infection. Our data revealed differential gene expression of inflammatory cytokines, chemokines, Toll-like receptors, interferon (IFN)-regulatory factors (IRFs) and IFN-stimulated genes (ISGs) in PRRSV-infected macrophages. We also examined the expression profile of miRNA and tRNA in whole blood from PRRSV-infected and non-infected animals to investigate possible regulation of host gene silencing by the virus that could influence the function of host immune, metabolic, and structural pathways. From these targets we have characterized the replication-competent expression of interferons (IFNs) using a PRRSV infectious clone in terms of the viral replication kinetics, IFN expression and stability. Collectively, these studies help set the stage for development of novel therapies and vaccine strategies. Several novel vaccine candidates were compared to a commercial PRRSV vaccine and found to be as effective or better at reducing viral load and fever following a field-isolate PRRSV challenge. The United States Swine Pathogen Database was developed at the National Animal Disease Center (https://swinepathogendb.org). In support of Objective 3, the new database was designed for the exploration of carefully curated genetic sequences to allow researchers and stakeholders to determine how genetic diversity of swine pathogens is changing spatially and temporally. It is comprised of nucleotide sequences and related metadata found in GenBank (part of the United States National Center for Biotechnology Information), and those nucleotide sequences and metadata detected by key veterinary diagnostic laboratories - Iowa State University Veterinary Diagnostic Laboratory (ISU VDL), the Kansas State University Veterinary Diagnostic Laboratory (KSU VDL), and the South Dakota Animal Disease Research and Diagnostic Laboratory at South Dakota State University (SD ADRDL). The database is currently housing data for porcine reproductive and respiratory syndrome virus (PRRSV), Senecavirus A (SVA), and porcine epidemic diarrhea virus (PEDV). A suite of web-based tools allows stakeholders to search for genetic sequence information, identify viruses similar to those circulating in their swine herd, and browse virus genomes to inform research and control efforts. The United States Swine Pathogen Database will also be used by veterinarians and researchers to study the evolution of those diseases most important to the United States swine industry. In support of Objective 4, we conducted studies investigating the pathogenesis, ecology and protective immune response of Senecavirus A (SVA), a swine virus that emerged as a problem for U.S. pork producers in 2015. Results from these studies demonstrated: 1) SVA could cause vesicular lesions in swine; 2) Clinical disease could be induced with “old” SVA isolates as well as contemporay isolates; 3) Wild-type SVA infection can induce a protective immune response with a duration of at least 4-5 months; 4) SVA transmission can occur for at least 2 weeks post infection to age-matched sows; 5) Environmental contamination may be a likely source of SVA detected in sows moving from farm to eventual slaughter; and 6) Demonstrated an inactivated whole-virus SVA could be used as a vaccine to prevent clinical disease in vaccinated swine. This information will help in developing response strategies at slaughter houses which can help in developing control programs on the farm. In response to the COVID-19 pandemic and concerns about animals serving as a virus reservoir for people, we collaborated with scientists at Tennessee State University, Nashville, Tennessee to evaluate cross-species ACE2 genetic (and especially epigenetic) diversity in regulation of ACE2 expression (the virus receptor on the host cell) to determine the cell tropism and animal susceptibility to SARS-CoV2. In addition, animal studies were conducted in collaboration with scientists from Cornell University, Ithaca, New York to evaluate the susceptibility of swine, cattle, and white-tailed deer to infection with SARS-CoV-2. White-tail deer were shown to replicate SARS-CoV-2 and transmit the virus to contact deer. In contrast, swine and cattle did not have sustained replication of the virus and seem unlikely to serve as a SARS-CoV-2 reservoir. Studies were completed to evaluate/improve diagnostic assays that included collaboration with scientists in the APHIS Diagnostic Virology Laboratory (DVL) in Ames, Iowa as well as with several state veterinary diagnostic laboratories associate with state universities. One animal study analyzed the antibody response of swine to the recently emerging Lineage 1C PRRSV 1-4-4 isolates that are causing concern for U.S. pork producers and our trading partners. Serum reagents from this study demonstrated antibodies against this new lineage could be detected with current diagnostic assays utilized in DVL, thus trading partners should not be concerned. Studies were completed evaluating new diagnostic methods for the detection of pseudorabies virus and specific antibody which indicated these new methods were sensitive and specific enough to be incorporated into current diagnostic protocols which improved the response capacity of U.S. diagnostic laboratories.

Accomplishments
1. Demonstrated swine were not susceptible to SARS-CoV-2 infection. Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of the COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. ARS researchers in Ames, Iowa, evaluated the susceptibility of cattle, swine, and white-tail deer to SARS-CoV-2 virus. Cattle and pigs were not susceptible to a sustained infection demonstrating these species are unlikely to pose a risk to humans. White-tailed deer were infected, but did not develop clinical disease. However, the infected deer did shed infectious SARS-CoV-2 to close contact deer demonstrating efficient SARS-CoV-2 transmission. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus that could play a role in the epidemiology and spread of SARS-CoV-2 in the human population.

2. Development of the United States Swine Pathogen Database: a platform to integrate veterinary diagnostic laboratory sequence data to monitor emerging pathogens of swine. In recent years, several viral diseases of pigs have emerged in the United States causing hundreds of millions of dollars in economic damage. To effectively respond to these diseases or detect new disease incursions or viral variants, it is critical to have a database of currently circulating viral genetic sequences and associated tools to analyze the sequences. ARS scientists in Ames, Iowa, collaborated with scientists at Iowa State University, Kansas State University, South Dakota State University, and Cornell University to establish the United States Swine Pathogen Database. The database houses genome sequences (both from the diagnostic laboratories and from GenBank) for porcine reproductive and respiratory syndrome virus, Senecavirus A, porcine epidemic diarrhea virus, and African and Classical swine fever viruses, and Foot-and-mouth disease virus nucleotide sequence and related metadata (such as US State and date of sample collection) parsed from public resources and previously private clinical cases from veterinary diagnostic laboratories. A suite of web-based tools allows stakeholders, researchers, and veterinarians to quickly search for genetic sequence information, identify similar viruses, and browse virus genomes to inform their research and control efforts. This publicly available database (https://swinepathogendb.org/) will greatly increase researchers’ understanding of endemic circulating viruses and speed response efforts by helping them to quickly identify new viral variants.

3. Demonstrated the persistence of atypical porcine pestivirus (APPV) in swine. In piglets, APPV has been associated with congenital tremors (CT) which is recognized as muscle tremors that can be severe enough that piglets are unable to suckle. Experimental studies have reported presence of the virus throughout various tissues in affected piglets, but there is limited information available about cellular location of the virus in various tissues. In addition, other pestiviruses have been reported to persist in animals even after clinical signs have resolved, so questions remain about the persistence of APPV in tissues. ARS researchers in Ames, Iowa, demonstrated a broad cell distribution of APPV viral RNA in infected pigs including endothelial cells, fibroblasts, smooth muscle, and brain. RNA presence in tissues was more pronounced in acutely infected neonatal piglet tissues compared to recovered boars (~11 months-of-age), with the notable exception of diffuse presence of RNA in the cerebellum in boars. Results from this study have better characterized location of APPV in tissues of acutely affected pigs. In addition, the presence of APPV in boar tissues well after resolution of clinical signs suggests persistence of the virus similar to other pestiviruses. Persistence of APPV without apparent clinical signs could be contributing to the spread of APPV in the swine industry and further research should be directed at measuring the duration of APPV shedding in persistently infected animals to help develop measures for APPV control and prevention.

4. Improved diagnostic assays for detection of pseudorabies virus (PRV) in oral fluids. PRV infection can produce severe disease in pigs and significant economic loss to producers. PRV has been eradicated from the U.S. domestic swine herd, but the virus can be found in some wild pig populations making ongoing PRV control a challenge. Improving PRV control and detection involves rapid testing of large groups of swine, which can be done by testing oral fluid samples for the virus. ARS researchers in Ames, Iowa, conducted a PRV challenge study to produce oral fluids that were tested by a polymerase chain reaction (PCR) test. Results indicated the PCR test for PRV worked similarly for both oral fluids and the more traditional nasal swab sample; therefore, using oral fluids combined with the new test could be a useful tool for PRV surveillance and detection. This will benefit veterinarians and pork producers as they improve rapid response plans for PRV control.

5. Evaluated the difference between Senecavirus A (SVA) isolates collected during the 2015 outbreak to those isolated prior to 2015. SVA is a picornavirus that causes vesicular disease, or blister-like lesions, in swine. SVA was first discovered as a cell culture contaminant in 2002, but subsequent analysis determined that the National Veterinary Services Laboratory had found similar isolates in swine samples since the late 1980s. Attempts to reproduce clinical disease experimentally in swine with historical isolates were unsuccessful. After outbreaks of vesicular disease in the U.S. in 2015, multiple research groups were able to reproduce vesicular disease in swine with contemporary isolates. Due to this discrepancy, it was hypothesized that SVA isolates prior to 2015 were less pathogenic than contemporary isolates. ARS researchers in Ames, Iowa, compared the pathogenicity of three historical (2002, 2011, 2012) and three contemporary (2015) SVA isolates in growing pigs. All isolates produced similar clinical disease, however shedding dynamics were different among the isolates. All animals developed antibodies that would react against each of the viruses used in this study. This research demonstrated that both historical and contemporary SVA isolates could cause vesicular disease in swine in contrast to previous attempts with historical isolates. In addition, this study provided evidence that antibodies generated from an SVA vaccine should be protective against any SVA strains that could be currently circulating in the U.S. swine herd. Veterinarians and pork producers may use this information to design SVA control procedures.

6. Developed an infectious clone of porcine deltacoronavirus (PDCoV) to enable studying how this virus infects pigs. PDCoV is an enteric pathogen of swine that can cause diarrhea, dehydration, and mortality, especially in neonatal swine. ARS scientists in Ames, Iowa in collaboration with scientists at Loyola University produced full-length PDCoV cDNA which was transcribed in vitro and translated by host cells to yield infectious virus. This recombinant virus (icPDCoV) was compared to the wild-type PDCoV in neonatal piglets. The recombinant virus produced clinical signs that were indistinguishable from the wild-type virus. IT can now be utilized to generate vaccine candidates that have been modified to alter specific regions of the virus that have been shown to suppress the host immune response, a key to generating an effective vaccine. Efficacious vaccine candidates could be utilized to reduce morbidity and mortality associated with PDCoV infection in swine and assist in the control and prevention of PDCoV in swine.

7. Development of a common open-source infrastructure framework to improve management of community databases. Open access databases serve as central repositories for research communities to store, find, and analyze integrated, multi-disciplinary datasets. With increasing volumes, complexity, and the need to integrate disparate data types, community databases face tremendous challenges in ongoing maintenance, expansion, and upgrades. To solve this problem, scientists at the University of Tennessee, University of Saskatchewan, Washington State University, University of Connecticut, Biodiversity International, and ARS scientists from Ames, Iowa, and Beltsville, Maryland, developed a common database framework that implements best practices. The system, called Tripal, can reduce development burden, provide interoperability, ensure use of common standards, and is sustainable. The platform allows organisms, including swine pathogens, to be annotated to facilitate data mining and hypothesis generation. The integrated tools provide researchers timely access to sequences and associated descriptive data, allowing for biological data mining and epidemiological studies. These results allow for a better understanding of organisms stored in such databases, and for swine pathogens, Tripal is implemented to describe the emergence of novel viruses, how these novel pathogens are disseminated in the U.S. and abroad, and provides a toolkit for discovering new patterns of how viruses are moving through susceptible populations.

Review Publications
Fleming, D.S., Miller, L.C., Tian, Y., Li, Y., Ma, W., Sang, Y. 2020. Impact of porcine arterivirus, influenza B, and their coinfection on antiviral response in the porcine lung. Pathogens. 9(11). Article 9110934. https://doi.org/10.3390/pathogens9110934.
Sang, E.R., Tian, Y., Gong, Y., Miller, L.C., Sang, Y. 2021. Epigenetic evolution of ACE2 and IL-6 genes as non-canonical interferon-stimulated genes correlate to COVID-19 susceptibility in vertebrates. Genes. 12(2). Article 12020154. https://doi.org/10.3390/genes12020154.
Deng, X., Buckley, A.C., Pillatzki, A., Lager, K.M., Faaberg, K.S., Baker, S.C. 2020. Inactivating three interferon antagonists attenuates pathogenesis of an enteric coronavirus. Journal of Virology. 94(17). https://doi.org/10.1128/JVI.00565-20.
Buckley, A.C., Michael, D.D., Faaberg, K.S., Guo, B., Yoon, K., Lager, K.M. 2020. Comparison of historical and contemporary isolates of Senecavirus A. Veterinary Microbiology. 253. Article 108946. https://doi.org/10.1016/j.vetmic.2020.108946.
Deng, X., Buckley, A.C., Pillatzki, A., Lager, K.M., Baker, S.C., Faaberg, K.S. 2020. Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026. Virology. 553:35-45. https://doi.org/10.1016/j.virol.2020.11.002.
Buckley, A.C., Falkenberg, S.M., Palmer, M.V., Arruda, P.H., Magstadt, D.R., Schwartz, K.J., Gatto, I., Neill, J.D., Arruda, B.L. 2021. Distribution and persistence of atypical porcine pestivirus (APPV) in experimentally inoculated pigs. Journal of Veterinary Diagnostic Investigation. 33(5):952-955. https://doi.org/10.1177/10406387211022683.
Palmer, M.V., Martins, M., Falkenberg, S.M., Buckley, A.C., Caserta, L.D., Mitchell, P.K., Wagner, B., Cassmann, E.D., Lager, K.M., Diel, D.G. 2021. Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2. Journal of Virology. 95(11). Article e0008321. https://doi.org/10.1128/JVI.00083-21.
Cheng, T., Henao-Diaz, A., Poonsuk, K., Buckley, A.C., Van Geelen, A., Lager, K.M., Harmon, K., Gauger, P., Wang, C., Ambagala, A., Zimmerman, J., Gimenez-Lirola, L. 2021. Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR. Preventive Veterinary Medicine. 189. Article 105308. https://doi.org/10.1016/j.prevetmed.2021.105308.
Falkenberg, S.M., Buckley, A.C., Laverack, M., Martins, M., Palmer, M.V., Diel, D., Lager, K.M. 2021. Experimental innoculation of young calves with SARS-CoV-2. Viruses. 13(3). Article 441. https://doi.org/10.3390/v13030441.