Detection and genetic characterization of emerging and re-emerging viral enteric diseases in swine in Thailand

Abstract

Emerging and re-emerging important enteric viruses in pigs including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and enterovirus G (EVG) are important pathogens of food security and public health concerns. In Thailand, the information on the occurrences and status of PEDVs, PDCoVs and EVGs is limited. Especially, Thai-EVGs have never been reported before. This thesis composes of three study phases. Phase 1 is surveillance of swine enteric viruses in pig farms. Phase 2 is genetic characterization and phylogenetic analysis of swine enteric viruses. Phase 3 is development of rapid diagnostic tests using RT-LAMP with lateral flow device (LFD) and DNA aptamer. For phase 1, surveillance of swine enteric viruses in pig farms was performed during December 2014 – January 2018. The fecal and intestinal samples (n=777) were collected from 73 pig farms from 20 provinces of 7 livestock regions. The occurrences of PEDVs, PDCoVs and EVGs by samples were 44.02%, 3.47% and 71.56%, respectively. By pig farms, the occurrences of PEDVs, PDCoVs and EVGs were 50.68%, 9.59% and 69.86%, respectively. Thai-PEDVs and Thai-EVGs were circulating throughout the country. While Thai-PDCoVs were only circulating in high density of pig production provinces of Thailand. Age groups of pigs associated with Thai-PEDVs and Thai-EVGs infections and seasonal patterns associated with Thai-PDCoVs and Thai-EVGs infections were observed. Moreover, the co-circulation of PEDVs, PDCoVs and EVGs with low rate (0.13%) were detected. For phase 2, for Thai-PEDVs, representative PEDVs (n=39) were classified into 3 genotypes (Novel G1, G2a and Novel G2). While Novel G1 and Novel G2 have never been reported in Thailand before. At least 3 epitopes (COE, SS6 and 2C10) showed multiple amino acid changes and 2 novel patterns at epitopes SS6 (764PQEGQVKI771) and 2C10 (1368GPRFQPY1374) were identified. For Thai-PDCoVs, representative PDCoVs (n=16) were grouped into Thailand cluster which closely related to Laos-PDCoVs. The multiple amino acid substitutions at 3 epitopes (NTD, CTD and S2) were observed. For Thai-EVGs, representative EVGs (n=34) were classified into 6 genotypes (G1, G3, G4, G8, G9 and G10) which the predominant genotype of Thai-EVGs was G3. The age groups of pigs were associated with genotypes of EVGs infection. For phase 3, The RT-LAMP with LFD kits with high sensitivity and specificity were developed to differentiate PEDVs and PDCoVs infections in field settings. The 2 candidate aptamers (N04 and N25) which had specific binding and high binding affinity to NP protein of PEDV were established. In summary, our results confirmed that swine enteric viruses are circulating in pig farms in Thailand. Therefore, the results of surveillance of swine enteric viruses in pig farms and genetic characterization of swine enteric viruses provided valuable information for prevention and control of swine enteric viruses. Moreover, the rapid diagnostic kits could be applied for early detection and distinguish between PEDVs and PDCoVs infections.