Project : USDA ARS

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

Location: Virus and Prion Research

2024 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
With the continued circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the globe, concerns about animals serving as reservoirs for the virus remains with the potential to contribute to the ecology and epidemiologic spread of the virus in the human population. Research efforts from this unit were redirected to help investigate these concerns by providing coronavirus expertise. Our research unit was a critical member of the first group to demonstrate that white-tailed deer were susceptible to SARS-CoV-2 infection and could transmit the virus to naïve deer. Subsequent research has documented the pathogenesis of the virus in both adolescent and adult white-tailed deer. Interestingly, deer do not show clinical signs after infection, though they can shed live virus in bodily secretions for approximately 1 week. Follow-up work has also demonstrated long lasting neutralizing antibodies at least 8 months after inoculation. Interest in assessing other wildlife species that may be susceptible to SARS-CoV-2 infection led our team to aid in research with both North American elk and bison. Results from these studies have shown that both species do not replicate the virus well; although, both mounted an immune response after inoculation. This work is critical to improving our understanding of the role animals may play in the epidemiology and spread of SARS-CoV-2 in wildlife as well as the possibility of spread to the human population. In support of Objective 2, a collaboration was developed with other ARS researchers to study the role of various receptors in the susceptibility of swine to different swine coronaviruses. Developing pigs with resistance to swine coronavirus infections could help control and/or prevent endemic as well as emerging coronaviruses in the swine industry. Initial in vitro work utilized primary cell lines with a known receptor for porcine epidemic diarrhea virus (PEDV) knocked out and these cells were infected with PEDV. The cells did not become infected; therefore, an animal study was initiated to see if these results could be repeated in vivo. Both knockout and wildtype pigs were able to replicate and shed PEDV, thus demonstrating that PEDV utilizes more than one receptor to infect swine. Follow up work may target additional receptors and include challenge with other swine coronaviruses. In support of Objective 3, a study was performed to characterize the pathogenesis of porcine circovirus 3 (PCV3), as well as test the efficacy of a protein subunit vaccine. Although PCV3 was discovered approximately 10 years ago there is still much unknown about its role as a primary pathogen in swine as reliable animal models have not been established and this agent is ubiquitous in swine facilities. Previously published research used immune suppression to replicate disease. In our work, we were able to get viral replication in cesarean-derived colostrum-deprived (CDCD) pigs without the use of immune suppression. In addition, this study was able to characterize the cellular and humoral immune response of both vaccinated and unvaccinated animals after challenge. This work improves understanding of the pathogenesis of PCV3 in swine as well as a possible vaccine approach to control the spread of this virus in the swine industry. Senecavirus A (SVA) is a causative agent for vesicular disease in swine and there is currently no commercial vaccine available for control measures. Previous research by this lab demonstrated that one dose of an SVA whole-virus inactivated vaccine developed in house was able to reduce the development of vesicular lesions. To model a more readily available vaccine approach in the swine industry, we tested the efficacy of an autogenous vaccine generated by a commercial supplier. Results from this study demonstrated that two-doses of the autogenous vaccine did not generate a robust neutralizing antibody response in swine; however, fewer animals in the vaccinated group developed vesicular lesions compared to the non-vaccinated animals. Since SVA causes clinical disease identical to foot-and-mouth disease, which is a reportable disease, control and prevention measures are critical to reducing the number of foreign animal disease investigations that must be performed every time a vesicle is observed. Finally, we collaborated with multiple universities to investigate the pathogenesis of an emerging respiratory pathogen in the swine industry, porcine astrovirus 4 (PoAstV4). Although this virus had been detected in respiratory cases in swine, its role as a primary cause of respiratory disease had not been demonstrated. An animal study was performed with CDCD piglets to characterize the course of infection and provide positive samples to be utilized to validate diagnostic assays for the swine industry. No clinical respiratory signs were observed after inoculation, though nasal swabs were able to demonstrate viral replication. This work suggests that PoAstV4 may not be a primary respiratory pathogen and could require other co-factors to cause respiratory disease.

Accomplishments
1. Demonstrated the efficacy of a novel replication-competent recombinant Porcine Reproductive and Respiratory syndrome virus (PRRSV) modified live vaccine (MLV). PRRSV is the most economically costly disease impacting the United State swine industry. There are currently commercial vaccines available; however, it can be a challenge for vaccines to provide protection against the wide viral diversity of circulating PRRSV isolates. ARS researchers in Ames, Iowa, developed an MLV that also expressed the potent antiviral cytokine interferon (IFN) omega5 to combat PRRSV infection. An animal challenge study was performed that assessed the efficacy of this novel MLV in comparison to a commercial MLV. The IFN-omega MLV group showed enhanced early immune activation compared to traditional MLV vaccines and underscores the potential that IFN-omega5 expression has as a promising strategy for developing more effective PRRSV vaccines with broader coverage against diverse PRRSV challenges. These results advance efforts towards effectively controlling and preventing PRRSV infections to mitigate its economic and health impacts on pig production and the swine industry.

2. Exposure to SARS-CoV-2 did not protect mink from reinfection with either the same or different SARS-CoV-2 variant. Mink were one of the first animals to be impacted by the SARS-CoV-2 pandemic with mink farms reporting mortality and morbidity in multiple countries. Concerns over mink contributing to the spread and mutation potential of SARS-CoV-2 even lead to the depopulation of some farms. To better understand infection dynamics, best samples to collect for diagnosis, and the immune response to infection, ARS researchers in Ames, Iowa challenged two groups of mink with an ancestral Wuhan-like or Omicron variant of CoV-2 and then both groups were re-challenged 4 weeks later with the same Omicron variant. Nasal washes were the best individual sample to detect viral replication; however, swabs of the litter pan were sufficient for surveillance of farms and did not require handling individual animals. Mink produced high levels of non-neutralizing antibodies with lower levels of neutralizing antibodies which could help explain why both groups still shed virus in nasal secretions after re-challenge. This work will help guide mink producers with sampling recommendations for SARS-CoV-2 surveillance and testing. In addition, demonstration that wild-type exposure to SARS-CoV-2 does not provide immunity can provide an impetus to promote the importance of increased biosecurity measures on mink farms and precautions for animal caretakers around these animals.

3. Demonstrated that swine persistently infected with atypical porcine pestivirus (APPV) can transmit virus to naïve contact animals. APPV has been found to be associated with piglets demonstrating congenital tremors (CT), but also has been identified in swine of all ages without any clinical disease. Previous research suggested that APPV infection may persist in swine leading to infection of cohorts and to CT offspring. ARS researchers in Ames, Iowa, conducted a study following gilts with evidence of persistent virus in serum and oral fluids, to analyze transmission dynamics to naïve contacts as well as whether they would give birth to CT piglets. Gilts were able to transmit the virus to naïve contact animals throughout breeding and gestation and oral fluids from these gilts remained positive for the duration of the study, but upon farrowing these gilts did not produce any piglets with CT and did not infect their piglets. This study has shed light on infection dynamics of APPV in swine especially the duration of shedding of APPV in oral fluids. A better understanding of the duration of transmission can aide in developing control measures for the swine industry such as limiting swine movement if APPV has been detected on a farm to prevent spread of the virus.

U.S. DEPARTMENT OF AGRICULTURE

Virus and Prion Research: Ames, IA