DETECTION AND MOLECULAR CHARACTERISATION OF VIRUSES ASSOCIATED WITH REPRODUCTIVE DYSFUNCTION IN PIGS WITH SPECIAL REFERENCE TO PORCINE TESCHOVIRUS
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Date
2023-03-22
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Abstract
The study was undertaken to detect viruses associated with reproductive dysfunction in pigs in Kerala with special reference to porcine teschovirus (PTV). Apart from PTV, the other viruses tested were porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV). A total of 45 tissue samples and faecal samples from animals with history of enteritis and reproductive dysfunction were collected. Faecal samples were also collected from 30 live pigs in farms with history of enteritis and reproductive dysfunction. Total RNA/DNA were extracted separately from the tissue samples using RNA/DNA extraction kit. Total RNA was also extracted from fecal samples. Complementary DNA (cDNA) was synthesized from extracted RNA using cDNA synthesis kit. The cDNA was used as template in the reverse transcription polymerase chain reaction (RT-PCR) for detection of 5’UTR region of PTV. The results of the test revealed that 18 out of 45 samples were positive for the virus with positivity rate of 40 per cent. The positive samples were further subjected to RT-PCR with primer sets for detecting the VP1 gene of the virus. However, sequence was obtained and the genotyping of the virus could not be carried out. In order to obtain the complete VP1 sequence, the forward primer binding to 5’UTR region and reverse binding to VP1 region was used to amplify a 3.3 kb region of the virus. In this test, a specific amplicon was obtained in one of the samples. The complete 3.3 kb amplicon was sequenced by Nanopore single strand sequencing technology using MinION equipment. The obtained sequences were mapped to PTV1 reference sequence using Geneious Prime software. On analysis of the VP1 sequence thus obtained, and subsequent phylogenetic analysis, it was observed that the sequence was similar to VP1 sequence of PTV
14. Out of the samples tested, 11 out of 45 were positive in RT-PCR for detection of ORF6 of PRRSV with positivity of 24.44 per cent. The BLAST analysis of the ORF6 sequences of PRRSV revealed that the isolates were similar to isolates from China (96.86 - 99.21 per cent similarity) and Switzerland (98.60 per cent similarity). The PPV was detected in 4 positives out of 45 samples (8.89 per cent similarity). The BLAST analysis of the sequences revealed that the isolates were similar to isolates from Italy (100 per cent similarity), China (100 per cent similarity), Germany (100 per cent similarity), South Korea (100 per cent similarity) and Brazil (100 per cent similarity) while the sequences were similar
to isolates from Thrissur, Kerala only by 99.50 per cent. One of the animals had mixed infection with PTV and PRRSV and three animals had mixed infection with PPV and PTV. It is concluded that PTV is present at an alarming rate in pigs of Kerala. Also present are PRRSV and PPV, but to a lesser level compared to PTV. The presence of these viruses alone or in combination may be responsible for the reproductive dysfunction observed in pigs in Kerala.
Key words: Reproductive dysfunction, PTV, PRRSV, PPV, RT-PCR, MinION