The technique of RT-PCR and restriction enzyme analysis was standardized to detect and differentiate Newcastle disease viruses. Digestion of RT-PCR-amplified, F gene sequences encoding for the cleavage activation sites of fusion protein with restriction enzymes AluI, BglI, HaeIII, HinfI, HhaI, RsaI, StyI and TaqI was carried out in order to characterize Newcastle disease viruses of varying pathogenicity. Restriction enzyme digestion of the amplicons by BglI and HhaI could group eight viruses, both field isolates and known vaccine strains, into lentogenic, mesogenic and velogenic pathotypes. By employing this technique directly on a clinical sample, Newcastle disease virus of the lentogenic pathotype could be detected.
Five field isolates of Newcastle disease virus, including one from a pigeon from the Indian subcontinent, along with three vaccine strains have been characterized by sequence analysis of the fusion protein (F) gene in the region encoding the F 2 -F 1 cleavage site. Based on the amino acid sequence present at the cleavage site and on the percent divergence at nucleotide and amino acid levels, three field isolates could be classified as velogenic and two were of lentogenic pathotypes. The velogenic pathotypes had the sequence RRQK/RRF at the cleavage site, while the lentogenic strains had GRQA/GRL at the corresponding position.
A single-tube, non-interrupted, one-step RT-PCR has been standardized to amplify the hypervariable region of the VP2 gene sequence of infectious bursal disease virus (IBDV). The technique standardized on purified viral RNA was successfully applied to the detection of the virus directly in clinical samples. The amplified products were confirmed to be IBDV specific by their size in ethidium bromide-stained agarose gel, nested PCR and restriction enzyme digestion. Digestion of the amplicons with StyI restriction enzyme also differentiated classical virus from six very virulent field isolates. The sensitivity of the one-step RT-PCR was found to be 0.2 pg of viral RNA.
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