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

Location: Virus and Prion Research

2019 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
In support of Objective 1, a method for applying bioinformatics techniques to older, yet to be analyzed sequence data was developed. The data was obtained from experiments completed in Ames, Iowa, using tracheobronchial lymph nodes that were infected with either porcine reproductive and respiratory syndrome virus, swine influenza virus A, porcine circovirus type 2, or pseudorabies virus. This data was originally sequenced using an older tag-based sequencing method, Digital Gene Expression Tag Profiling (DGETP) (circa 2007). Using DGETP, a specific segment from each transcribed gene is recovered from the tissue under study, sequenced and counted to provide a transcription profile revealing what genes are transcribed and how often. These were then mapped to the current swine genome assembly to compare, contrast, and characterize genes and regulatory pathways that overlap the immune response to these swine viruses. In support of Objective 1, a recombinant porcine delta coronavirus virus (PDCoV) was produced in collaboration with scientists at Loyola University. Based on reported full-length PDCoV genomes, a recombinant genome was assembled but did not replicate in cultured cells. To understand the nucleotide differences in the recombinant virus that may have prevented replication in cell culture, it was compared to the original PDCoV field isolate genome which identified changes in the spike protein gene. A change in the spike gene was engineered into the recombinant PDCoV genome that allowed the recombinant PDCoV virus to grow in cell culture. In support of Objective 2, to assess the ability of multiple porcine reproductive and respiratory syndrome virus (PRRSV) strains to replicate together in cell culture, two recombinant differentiating infected from vaccinated animals (DIVA) vaccine viruses were produced that possessed a foreign tag in a modified-live virus (MLV) replicase region with structural regions of two contemporary PRRSV, which are termed chimeras. The two chimeric viruses were propagated alone or together in cell culture until cytopathic effect was observed and viral supernatants were processed and submitted for next generation genome nucleotide sequencing. Only one chimeric viral genome was detected in the mixed culture. The next experiments will assess the abilities of two chimeric viruses to replicate when a series of ratios between the two chimeras are evaluated in cell culture. The research will determine the ratio at which both chimeric viruses will be detectable in cell culture, to be used for preliminary studies in swine. In support of Objective 2, scientists at the National Animal Disease Center, Ames, Iowa, and Tennessee State University characterized the effect of replication-competent expression of interferons (IFNs) using a porcine reproductive and respiratory syndrome virus (PRRSV) infectious clone. Viral replication kinetics, IFN expression and stability, and acquired antiviral activity in infected cells, including porcine primary macrophages and monocyte-derived dendritic cells, were examined. As one major feature of the tested vaccine candidates that showed protection, the scientists hypothesized that the virus-introduced IFNs serve to reverse PRRSV’s suppression on endogenous IFN signaling and inducing antiviral immunity. The next step will be to characterize the in vivo responses during animal vaccine validation. In support of Objective 3, a new United States Swine Pathogen Database was developed at the National Animal Disease Center, Ames, Iowa (https://swinepathogendb.org). The database 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 laboratories. Presently, the South Dakota Animal Disease Research and Diagnostic Laboratory, the Iowa State Veterinary Diagnostic Laboratory and the Kansas State University Veterinary Diagnostic Laboratory have submitted over 2000 sequences of field submissions. 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 3, phylogenetic analysis of porcine reproductive and respiratory syndrome virus (PRRSV) was studied, using both open reading frame 5 (ORF5) and genomic nucleotide sequences of contemporary field samples sequenced by the South Dakota Animal Disease Research and Diagnostic Laboratory, the Iowa State Veterinary Diagnostic Laboratory and the Kansas State University Veterinary Diagnostic Laboratory as well as those found in GenBank, part of the United States National Center for Biotechnology Information. Key findings revealed that PRRSV isolates detected in Asia predominantly cluster into different lineages than those detected in the United States. However, limited numbers of PRRSV field samples with nucleotide similarity to United States strains NADC30 and NADC34 have recently been discovered in China. Peru has also identified field samples with nucleotide similarity to NADC34. The data suggests that the United States viruses are being transported abroad. In support of Objective 4, an animal study was completed that demonstrated Senecavirus A (SVA) is distributed throughout the pig’s tissues during the first week after infection with SVA. The tissues with the highest levels of viral nucleic acid by polymerase chain reaction (PCR) assay were the tonsils and lymph nodes. The next experiment will compare the temporal tissue distribution of a more contemporary SVA isolate (2015 versus 2018) which may provide insight to the purported differences in clinical disease seen in the field.

Accomplishments
1. Characterized the pathogenesis of Senecavirus A (SVA) in market-weight gilts. After ARS researchers in Ames, Iowa, demonstrated that SVA was a causative agent for vesicular disease in nursery-aged-pigs, the next step was repeating the study with more mature animals because most animals affected by the sudden increase of vesicular disease cases in swine in the United States due to SVA in 2015 were market-weight animals headed to slaughter and sows/gilts in farrowing facilities. An experimental SVA infection study of market-weight gilts was conducted. One difference noted in older animals when compared to younger animals was the presence of more snout lesions. In addition, foot lesions were observed earlier in some older animals compared to younger animals. A better understanding of disease progression in swine through experimental studies is critical to helping producers identify vesicular disease in animals in the field. This is crucial since vesicular disease caused by SVA is indistinguishable from clinical disease caused by foot-and-mouth disease virus (FMDV). Currently FMDV is not found in the United States. If FMDV did enter the country there would be severe economic ramifications; therefore, producers must remain diligent and it is important to understand the ecology of SVA in United States swine farms.

2. Identified the effect that porcine reproductive and respiratory syndrome virus (PRRSV) infection has on the display of activated monocytic cell signature genes. Monocytic cells are one of the blood cell types that are intricately involved in the animal's response to disease. They have a role in the innate immune response as well as in development and maintenance of adaptive immunity (the basis for vaccination) against invading pathogens. Understanding the immunological impact is increasingly important for investigating host-virus interactions of existing and emerging pathogens. ARS scientists in Ames, Iowa, conducted extensive genome-wide profiling of signature genes in activated porcine monocytic innate immune cells. The data revealed differential expression of various genes involved in communication between cells of the immune system that are critical for development of protective immunity. This can help set the stage for development of novel therapies and vaccine strategies.

3. Evaluated the differential expression of small non-coding RNAs of the host cell that may regulate gene silencing in porcine reproductive and respiratory virus (PRRSV) infected animals. It has been established that reduced susceptibility to PRRSV has a genetic component that may take the form of small non-coding RNA (sncRNA) molecules that function as regulators of host and viral gene expression. In order to identify differences in sncRNA expression between healthy and highly pathogenic PRRSV (HP-PRRSV) challenged pigs, a transcriptomic analysis of porcine whole blood from control and infected pigs was examined for changes in expression profiles associated with the virus by ARS scientists in Ames, Iowa and at Tennessee State University. Results revealed multiple classes of sncRNA were both present and differentially expressed during HP-PRRSV infection. Transfer RNA fragments experienced a lower reduction in number than the microRNAs and appear to be more stable across time points than microRNA or other non-coding RNAs. This indicates the HP-PRRSV infection affects host homeostasis through changes in miRNA and tRNA expression and their subsequent gene interactions that target and influence the function of host immune, metabolic, and structural pathways. This information advances the understanding of how PRRSV can negatively affect the pig’s immune system which may aid in the development of improved PRRSV vaccines.

4. A new United States Swine Pathogen Database (https://swinepathogendb.org) was developed by ARS scientists in Ames, Iowa. The database 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 laboratories. Presently, the South Dakota Animal Disease Research and Diagnostic Laboratory, the Iowa State Veterinary Diagnostic Laboratory and the Kansas State University Veterinary Diagnostic Laboratory have submitted over 2000 sequences of field submissions. 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.

5. In collaboration with scientists at the Ohio State University, ARS scientists in Ames, Iowa performed an animal challenge study. The study describes the immune response of gilts exposed to porcine epidemic diarrhea virus (PEDV) at various time points prior to farrowing. Characterization of the immune response involved measurement of B cells, circulating PEDV antibodies, and antibody secreting cells. Another part of the immune response measured was lactogenic immunity by studying the protection colostrum and milk antibodies provided to piglets when challenged with PEDV. Piglets born to gilts exposed to PEDV during the second trimester of gestation had 100 percent survival rate when challenged with PEDV. On the other hand, piglet survivability was 87.2 percent and 55.9 percent for litters born to gilts exposed in the first and third trimester, respectively. This work clarifies the optimal timing for gilt exposure to provide ideal lactogenic immunity against PEDV infection.

Review Publications
Bester, S.M., Daczkowski, C.M., Faaberg, K.S., Pegan, S.D. 2018. Insights into the porcine reproductive and respiratory syndrome virus viral ovarian tumor domain protease specificity for ubiquitin and interferon stimulated gene product 15. ACS Infectious Diseases. 4(9):1316-1326. https://doi.org/10.1021/acsinfecdis.8b00068.
Buckley, A., Kulshreshtha, V., van Geelen, A., Montiel, N., Guo, B., Yoon, K., Lager, K. 2019. Experimental Seneca Valley virus infection in market-weight gilts. Veterinary Microbiology. 231:7-10. https://doi.org/10.1016/j.vetmic.2019.02.019.
Langel, S.N., Paim, F.C., Alhamo, M.A., Buckley, A., van Geelen, A., Lager, K.M., Vlasova, A.N., Saif, L.J. 2019. Stage of gestation at porcine epidemic diarrhea virus infection of pregnant swine impacts maternal immunity and lactogenic immune protection of neonatal suckling piglets. Frontiers in Immunology. 10:727. https://doi.org/10.3389/fimmu.2019.00727.
Buckley, A., Kulshreshtha, V., van Geelen, A., Montiel, N., Guo, B., Yoon, K.-Jin, Lager, K. 2019. Experimental Seneca Valley virus infection in market-weight gilts. Veterinary Microbiology. 231:7-10. https://doi.org/10.1016/j.vetmic.2019.02.019.
Buckley, A., Montiel, N., Guo, B., Kulshreshtha, V., van Geelen, A., Hoang, H., Rademacher, C., Yoon, K.-J., Lager, K. 2018. Dexamethasone treatment did not exacerbate Seneca Valley virus infection in nursery-age pigs. BMC Veterinary Research. 14:352. https://doi.org/10.1186/s12917-018-1693-8.
Shi, P., Su, Y., Li, R., Zhang, L., Chen, C., Zhang, L., Faaberg, K., Huang, J. 2019. Dual regulation of host TRAIP post-translation and nuclear/plasma distribution by porcine reproductive and respiratory syndrome virus non-structural protein 1alpha promotes viral proliferation. Frontiers in Immunology. 9:3023. https://doi.org/10.3389/fimmu.2018.03023.
Deng, X., van Geelen, A., Buckley, A.C., O'Brien, A., Pillatzki, A., Lager, K.M., Faaberg, K.S., Baker, S.C. 2019. Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type I and type III interferon responses. Journal of Virology. 93(8):e02000-18. https://doi.org/10.1128/JVI.02000-18.
Fleming, D.S., Anderson, S.J., Miller, L.C. 2018. Identification of coding and non-coding RNA classes expressed in swine whole blood. Journal of Visualized Experiments. (141):e58689. https://doi.org/10.3791/58689.