Assessment of different gene targets for detection of Peste des Petits ruminant virus by RT-PCR and sequence analysis of F and N gene segments
Nagaraj, Kerur (2005) Assessment of different gene targets for detection of Peste des Petits ruminant virus by RT-PCR and sequence analysis of F and N gene segments. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Peste des petits ruminants (PPR) is an acute viral disease of goats and sheep characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis and pneumonia. Goats are usually more severely affected than sheep. PPR is caused by Peste des petits ruminants virus (PPRV) a paramyxovirus of the Morbillivirus genus. In India, PPR was first reported in 1987 from Tamil Nadu and for several years, the disease was thought to be restricted to southern India only, however in 1994, a series of PPR outbreaks were reported from many northern states as well as from West Bengal. The disease causes severe losses to small ruminant production 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 sandwich-ELISA and to derive estimates of overall, locationwise and species wise incidence of PPRV. In addition to designing a new pair of N gene based primers, the study also involved the assessment of suitability of different gene targets for detection of PPRV by RT-PCR from the clinical samples. Further, an attempt was also made to characterize the local field isolates by cloning and sequencing the F and N gene fragments amplified by PCR. A total of 48 clinical samples from 32 animals from selected areas of Gujarat were tested by sandwich ELISA. Twenty six out of 32 animals tested were found positive for PPRV yielding an over all incidence rate of 81.25 per cent. Samples from all the animals in Mehsana, Banasantha and Patan districts of North Gujarat as well as one animal from Rajkot district of Saurashtra region were positive by sandwich ELISA, yielding 100 per cent incidence rates at these locations. However, incase of Anand district, four (40%) out of ten tested animals were positive. The incidence rates of PPR did not vary significantly between goats (80.00%) and sheep (83.33%). The PPRV antigen could be detected in more number of blood samples (66.66%) than in nasal swabs (40.00%), in cases where both sample types were taken from a same animal. The same 48 clinical samples were tested by the conventional F gene based RT-PCR and estimates of relative sensitivity and specificity were derived. When compared to sanwich ELISA (33 samples positive), the PPRV could be detected in fewer 24 (50.00%) of samples by the conventional F gene RT-PCR. Thus relative sensitivity and specificity of F gene based RT-PCR was 60.60 and 73.33 per cent respectively. The overall agreement between the two tests was 64.58 per cent. Further, three N gene based primers including a set of newly designed primer (N1/N2) and conventional F gene based primers were assessed for their efficacy in detecting the PPRV in clinical samples. The comparative analysis revealed that the N gene based primers NP3/NP4 and N1/N2 could detect the PPRV in more number of samples (12) than the primers pprn_fr2/ pprn_rev (zero) and F1/F2 (9). Thus, the N gene based primers (NP3/NP4 and N1/N2) were more sensitive than the F gene based primers in detection of PPRV in field samples, in addition, the NP3/NP4 and N1/N2 were equally efficient in detecting PPRV from the clinical samples. The PCR products generated by F1/F2 and N1/N2 primers were cloned and nucleotide sequences of the inserts were determined, to characterize the local field PPRV by analyzing the sequences with bioinformatics tools. Based on the sequence identity analysis, it was found that the viruses from distant geographical locations differed to a greater extent in their N gene sequences than in F gene sequences. This finding was supported by the branching pattern shown by PPRV in Phylogenetic tree based on respective gene sequences. The Phylogenetic tree based on the lineage specific F gene clustered all the Indian isolates including the isolates of Gujarat together into lineage 4, along with the isolates of Turkey, Iran and Pakistan. However, the Phylogenetic tree based on the 425 bp N gene sequences (amplified by N1/N2) revealed a different pattern of branching, clustering all isolates of India into a separate branch from Turkey and Nigerian Isolates. In addition, the branching pattern as revealed by network analysis was in conformity with the one produced by PHYLIP programme.
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