Location: Virus and Prion ResearchTitle: Inactivating multiple coronavirus interferon antagonists attenuates pathogenesis of porcine epidemic diarrhea virus
|DENG, XUFANG - Loyola University|
|PILLATZKI, ANGELA - South Dakota State University|
|BAKER, SUSAN - Loyola University|
Submitted to: American Association of Swine Veterinarians Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 9/23/2020
Publication Date: 2/18/2021
Citation: Buckley, A.C., Deng, X., Pillatzki, A., Lager, K.M., Faaberg, K.S., Baker, S. 2021. Inactivating multiple coronavirus interferon antagonists attenuates pathogenesis of porcine epidemic diarrhea virus [abstract]. American Association of Swine Veterinarians Annual Meeting. Poster No. 37.
Technical Abstract: Introduction Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes diarrhea and mortality in neonatal pigs. Available efficacious vaccines to prevent PEDV infection are limited and many producers continue to use feedback to boost farm immunity, which can carry the risk of unintentional exposure of animals to other agents. The objective of this research was to create an alternative PEDV vaccine candidate by attenuating the virus. Attenuation in this study was focused on inserting mutations into proteins known to block host interferon responses also known as interferon antagonists. Materials and Methods An infectious clone of wild-type PEDV (icPEDV-WT) was previously generated and used to engineer a recombinant virus with four mutations (icPEDV-mut4). The four mutations were located in 3 proteins: nonstructural proteins 1, 15, and 16. Sequencing was performed to confirm mutations. Growth kinetics of both viruses were compared on Vero and PK1 cell lines and interferon gene expression was measured by RT-qPCR . Thirty four piglets were weaned between 3-6 days of age and assigned to 3 treatment groups: icPEDV-WT (n=13), icPEDVmut4 (n=13), and controls (n=8). Piglets were inoculated orally with 2 mL of virus solution with a titer of 500 TCID50/pig or sham media. Piglets were rectal swabbed and given a clinical diarrhea score on 0-10, 12, 14, 18, and 21 days post inoculation (dpi). Clinical diarrhea scores were assigned with the following criteria: 0 = normal feces, 1 = soft stool, 2 = semi-liquid stool, 3 = liquid feces, 4 = voluminous watery diarrhea. Serum was collected on 0 and 21 dpi. Two piglets were euthanized on 2 dpi for collection of jejunum and ileum tissue samples and the remaining animals were euthanized on 21 dpi. Swabs were tested by RT-qPCR for the presence of virus and serum tested for IgG and neutralizing antibodies. Structural damage and virus location in tissues were analyzed by H&E and immunohistochemistry (IHC) respectively. Viral sequencing for the regions of site-specific mutation was completed on fecal swab RNA from late in infection. Results Growth kinetics on interferon non-responsive Vero cells were similar between icPEDV-mut4 and icPEDV-WT. In contrast, growth on interferon responsive PK1 cells of icPEDV-mut4 was reduced 1000-fold compared with wild-type, which would suggest that reduced interferon antagonism resulted in limited viral replication. This was supported by higher levels of interferons measured in icPEDV-mut4 infected PK1 cells compared to icPEDV-WT infected cells. All piglets inoculated with icPEDV-WT developed watery diarrhea and achieved a score of at least 3 for one day, while all piglets inoculated with icPEDV-mut4 had scores of 0 or normal feces for the entire study. Viral shedding quantified by RT-qPCR was consistently 100-1000 times lower in rectal swabs of icPEDV-mut4 infected animals. All challenged pigs developed an antibody response to infection, though icPEDV-mut4 piglets had lower IgG and neutralizing antibody levels compared to icPEDV-WT. Histology of intestinal sections from icPEDV-mut4 piglets showed no significant pathologic lesions. In contrast, icPEDV-WT piglets had classic microscopic lesions with villous atrophy. IHC detected viral antigen in icPEDV-WT tissue sections but not in icPEDV-mut4 tissues. Sequencing of the viruses over regions of mutation revealed that the mutations were stable. Conclusion Inactivating multiple interferon antagonists created an attenuated PEDV. Further research will need to evaluate the efficacy of this virus as a vaccine candidate.