ISOLATION OF PPR VIRUS, DETECTION BY ELISA AND DIFFERENT GENE BASED RT-PCR
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Date
2008
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AAU, Anand
Abstract
Peste des petits ruminants (PPR) is a severe viral disease of goats and sheep with high morbidity and sometimes high mortality characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis and pneumonia. PPR is caused by Peste des petits ruminants virus (PPRY), a Paramyxovirus of the Morbillivirus genus. The disease causes severe economic losses to small ruminant husbandry and is presently considered as one of the major threats to about 200 million small ruminant population of the country. The present study was aimed to detect PPRV in clinical samples using s-ELISA and to derive estimates of overall, locationwise and specieswise incidence of PPRV. The study also involved the isolation of PPR virus from clinical samples and assessment of F, N and H gene targets for detection of PPRV by RT-PCR from the clinical and cell culture samples. A total of 98 clinical samples comprising of 48 tissues, 12 blood and 38 serum samples from 79 animals including 26 sheep and 53 goats suspected of PPRV infection, were collected from three different districts of Gujarat (Rajkot, Bhavnagar and Gandhinagar) and tested for PPRV antigen by s-ELISA (PPR s-ELISA kit, developed by Division of Virology, IVRI, Mukteshwar), of which 75 animals were found positive yielding an overall incidence rate of 94.90 per cent. Out of 10 animals (goat) suspected of PPR, six were positive (60%) by s-ELISA in Rajkot district. All the 32 and 37 animals (sheep and goats) showing clinical signs suggestive of PPR from Bhavnagar and Gandhinagar districts were found positive by s-ELISA yielding 100 %'incidence at both these locations. Sheep was found to be more susceptible to infection yielding a positivity rate of 100 per cent than goats (92.4%). The PPR antigen could be detected marginally more in tissue samples (95.83%) than in blood (83.33%) and serum samples (89.47%) by s-ELISA. A total of 23 samples including 15 cell culture samples, six tissue samples, one whole blood and reference vaccine virus (Sungri isolate) were processed for RNA extraction using TRI ReagentĀ®. RNA samples showing acceptable purity and concentration were reverse transcribed with random hexamers to generate cDNA templates, which were subjected to PCR amplification using F, N and H gene based primers. Reference vaccine virus as well as 10, 11 and 8 out of 15 cell culture samples of the three isolates produced approximately 372 bp amplicon with F1 / F2, 351 bp amplicon with NP3 / NP4 and 713 bp amplicon with H1 / H2 primers respectively. A total of 23 samples were tested by both s-ELISA and RT-PCR, including reference vaccine virus, seven field samples (six tissues, one whole blood) and 15 cell culture samples. Out of the total samples tested, PPRV could be detected in reference vaccine virus and 19 samples by s-ELISA and 15, 18 and 11 samples including reference vaccine virus by F, N and H gene based RT-PCR respectively. None of the sample positive in RT-PCR was negative by s-ELISA. Relative to s-ELISA, sensitivity of the RT-PCR for F, N and H gene was 78.94, 94.70 and 57.89 respectively and specificity for all the three was 100 per cent. Three representative samples comprising of one pooled tissue sample, one whole blood sample and one serum sample were inoculated in Vero cells for five viral passages to isolate the field PPR virus. All the three samples inoculated showed similar CPE. During first passage, changes associated with rounding of cells and isolated initiation foci were observed. During second and third passages, CPE characterized by ballooning of cells and later aggregation of cells followed by formation of fusion mass and syncytia were recorded. Cell lysis was also observed in few cases. During fourth and fifth passages, the above mentioned CPE increased in intensity with more number of cells showing fusion mass and detachment. The monolayer infected with sterile PBS (negative control) did not show such changes. The isolation of PPRV in cell culture was confirmed by s-ELISA after second passage for all the three samples and after P3, P2 and P2 passage by F, N and H gene based RT-PCR.
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ANIMAL BIOTECHNOLOGY, A STUDY