Location: Virus and Prion Research
2023 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 2020 there were reports of morbidity and mortality of farmed mink, across the United States, that tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There was concern the mink farms could serve as a viral reservoir and a source of virus for human infections). Partial research efforts from this highly skilled unit were redirected to help investigate this concern. Several research groups around the world have experimentally challenged mink with SARS-CoV-2 and preliminary histologic reports have drawn comparisons between lesions observed in mink to those in humans suggesting mink could be a useful animal model for COVID-19. The objective of this work was to document the pathogenesis of contemporary SARS-CoV-2 isolates in mink. In addition, environmental samples were collected to provide insight on best and most convenient samples mink farmers could collect for SARS-CoV-2 surveillance. After challenge, very few respiratory clinical signs were observed and there were no sustained increases in body temperature. Weight loss, if present, was less than 10% of body weight. In the first week of infection, virus was detected in multiple tissues, but by the second week most samples collected were negative for SARS-CoV-2. Microscopic lesions were observed in the infected mink in both the upper and lower respiratory tract. Collectively, both an omicron and a delta SARS-CoV-2 variant could infect mink but did not cause severe disease in this trial. A better understanding of the pathogenesis of SARS-CoV-2 infection in mink could help define their role as a COVID-19 disease model and shape disease control measures for the mink industry.
In support of Objective 2, studies were continued utilizing air-liquid interface porcine respiratory epithelial cells (ALI-PRECs) to model virus-host interactions for swine viruses with the goal of characterizing the molecular host response to viral infections. To gain insight into the porcine immune response to coronaviruses, ALI-PRECs were inoculated with the neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) which primarily infects and replicates in the upper respiratory tract of swine. Preliminary results shed light on the mechanisms driving the immune response to PHEV, especially the interferon and chemokine responses involved in antiviral regulation against PHEV.
In support of Objective 3, we continued to conduct animal studies to characterize the pathogenesis of Senecavirus A (SVA) infection in swine. SVA is a causative agent for vesicular disease in swine. In addition, reports from the field have noted increases in neonatal mortality during some outbreaks on sow farms. The virus had been found in swine samples dating back to the late 1980s; however, there were outbreaks of the virus around the world starting in 2015 leaving questions about differences in pathogenicity between more historical and contemporary isolates. Previous research at NADC determined the minimum infectious dose of a 2011 SVA isolate in both neonates and market-weight pigs. To determine if there are any differences between historical and contemporary strains, a 2018 SVA isolate from a farm experiencing increased neonatal mortality was used in a replicate minimum infectious dose study in neonates. This study demonstrated that the 2018 SVA isolate had a similar minimum infectious dose to the 2011 isolate.
Atypical porcine pestivirus (APPV) has been found to be associated with piglets demonstrating congenital tremors (CT) and clinical presentation of CT in piglets was reproduced after experimental challenge. The virus has been identified in swine of all ages globally; although, little is known about the pathogenesis and epidemiology of APPV. Gilts born with CT and consistently tested positive for APPV in serum were selected at 6 months of age to study the duration of shedding and whether these animals would give birth to CT piglets. Contact pigs were placed at three different timepoints during gestation and at each timepoint at least one pig became infected with APPV. The gilts did not give birth to piglets with CT. This study has shed light on infection dynamics of APPV in swine and the duration of viral shedding.
Accomplishments
1. Demonstrated cattle are likely not susceptible to infection with Senecavirus A (SVA) eliminating a significant concern for the U.S. cattle industry. SVA causes vesicular disease in swine that is clinically indistinguishable from foot-and-mouth disease (FMD). FMD can cause vesicular disease not only in swine but also cloven-hooved animals. FMD is highly contagious and is on the World Organization for Animal Health’s list of reportable diseases. There would be significant economic repercussions if FMD were to be diagnosed in the U.S. since it currently is not found here. Recently, it was reported in Guangdong, China that buffalo experiencing clinical symptoms similar to FMD including mouth ulcers and lameness tested positive for SVA. This was the first report of SVA outside of swine. ARS researchers in Ames, Iowa, conducted a study to determine the susceptibility of cattle (Bos taurus) to SVA infection. Initial work demonstrated that bovine cell lines were susceptible to SVA infection. Subsequently, six colostrum-deprived Holstein calves were challenged with SVA intranasally. No vesicular lesions were observed after challenge. Serum, oral, nasal, and rectal swabs tested for SVA nucleic acid did not support significant viral replication and there was no evidence of seroconversion. Based on this work, cattle were not susceptible to experimental SVA infection, thus demonstrating to the cattle industry that SVA is not a significant concern currently and alleviating concerns about multiple vesicular disease agents in cattle.
2. Atypical porcine pestivirus (APPV) infection dynamics in swine from birth to market. APPV has been found to be associated with piglets demonstrating congenital tremors (CT) and clinical presentation of CT in piglets was reproduced after experimental challenge. Depending on the severity of the tremors, piglets can have a difficult time nursing, which can lead to morbidity and mortality. The virus has been identified in swine of all ages, globally; although, little is known about the pathogenesis and epidemiology of APPV. Likewise, the true incidence is not known. ARS researchers in Ames, Iowa, conducted a longitudinal study following two cohorts of pigs, those born in litters with piglets exhibiting CT and those born in litters without CT, to analyze the dynamics of APPV infection from birth to market. There was a wide range in the percentage of affected pigs within CT positive litters. Pigs from CT negative litters quickly developed viremia after exposure to the virus that was cleared after a couple months, and a majority seroconverted by the end of the study. In contrast, a greater percentage of pigs exhibiting CT remained PCR positive throughout the growing phase with only a few of these animals developing a detectable antibody response. APPV nucleic acid was present in multiple tissues from pigs in both groups at the time of marketing especially the cerebellum, nasal turbinate, and salivary gland. This study has shed light on infection dynamics of APPV in swine and the impact immune status and timing of infection have on the persistence of APPV in serum and tissues, which can aide control measures for the industry.
3. Identifying the role of host genetics in pseudorabies virus (PRV) infection status of feral swine to direct management strategies. Pseudorabies virus (PRV) is a herpesvirus and infection in swine that can result in central nervous system, respiratory, and reproductive clinical signs, which vary in severity with age at infection. PRV has been eradicated from the United States domestic swine herd, but the virus continues to circulate in feral swine populations. Feral swine populations and their distribution have expanded dramatically in recent years and are known reservoirs for PRV; thus, posing a sustained risk of disease spillover for domestic herds. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, ARS researchers in Ames, Iowa, conducted research to identify gene sets associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Gene sets identified in this study were associated with neuronal development, muscle cells and calcium transport, and lipid metabolic processes. Understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be readily applied to management strategies and reduce the risk of PRV exposure to the commercial swine population.
Review Publications
Li, J., Sang, E.R., Adeyemi, O., Miller, L.C., Sang, Y. 2022. Comparative transcriptomics reveals small RNA composition and differential microRNA responses underlying interferon-mediated antiviral regulation in porcine alveolar macrophages. Epigenetics. 13. Article 1016268. https://doi.org/10.3389/fimmu.2022.1016268.
Devries, A.C., Crawford, L., Hoffman, K., Falkenberg, S. 2022. Experimental Senecavirus A infection of bovine cell lines and colostrum-deprived calves. Viruses. 14(12):Article 2809. https://doi.org/10.3390/v14122809.
Wu, X., Hu, Y., Sui, C., Pan, L., Li, F., Miller, L.C., Lee, C., Cong, X., Li, J., Du, Y., Qi, J. 2022. Multiple site SUMOylation of FMDV 3C protease and its negative role in viral replication. Journal of Virology. 96(17). Article e061222. https://doi.org/10.1128/jvi.00612-22.
Wu, X., Chen, L., Siu, C., Hu, Y., Jiang, D., Li, D., Miller, L.C., Li, J., Cong, X., Lee, C., Du, Y. 2023. 3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. Virologica Sinica. 38(3):387-397. https://doi.org/10.1016/j.virs.2023.03.003.
Sarlo Davila, K.M., Sang, Y., Ma, W., Miller, L.C. 2023. Differential expression in the porcine thymus upon infection by PRRSV, Influenza B or their coinfection. World Congress of Genetics Applied in Livestock Production. p.3192-3195. Article 775. https://doi.org/10.3920/978-90-8686-940-4_775.
