RESUMO
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.
RESUMO
Degenerate primers based RT-PCR (previously described by [Avian Dis 26 (1997) 837]) has been used for the detection and differentiation of Newcastle disease (ND) viruses. Two sets of primers (A+B and A+C), with common forward primer and distinct reverse degenerate primers, designed from fusion protein gene encoding for cleavage site, could differentiate virulent and avirulent Newcastle disease viruses (NDV). Both sets of primers amplified "F" gene sequence of virulent (velogenic and mesogenic) viruses, whereas in avirulent strains, amplification was only with primer set A+C. Total 10 NDV isolates and two clinical samples including both known and unknown pathotypes, were checked. Based on amplification results 5 viruses were found to be virulent type and 6 as avirulent with one of the two clinical samples, earlier positive by RT-PCR using non-degenerate "F" gene specific primers was found negative in this study. The technique has been found to be a simple and quick for the detection and differentiation of virulent and avirulent NDV, which is important for control of the disease in the events of the outbreaks.
Assuntos
Galinhas , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Doenças das Aves Domésticas/virologia , Animais , Eletroforese em Gel de Ágar/veterinária , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/isolamento & purificação , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , VirulênciaRESUMO
The polypeptides of three fowl adenovirus-4 (FAV-4) field isolates of hydropericardium syndrome from various geographical areas of the country and the standard FAV-1 (CELO virus) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by protein immunoblotting with polyclonal antibodies to FAV-4 and FAV-1. Protein profile analysis of FAV-4 isolates revealed similarity of all the eight polypeptides with molecular weight ranging from 20 to 107 kDa but differed from CELO, particularly in their 24.2 kDa protein. Subsequent immunoblotting showed relatedness of at least five protein fractions of FAV-4 to CELO virus.
Assuntos
Infecções por Adenoviridae/veterinária , Galinhas/virologia , Adenovirus A das Aves/isolamento & purificação , Derrame Pericárdico/veterinária , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/virologia , Animais , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Adenovirus A das Aves/imunologia , Adenovirus A das Aves/metabolismo , Adenovirus A das Aves/patogenicidade , Derrame Pericárdico/patologia , Derrame Pericárdico/virologia , Doenças das Aves Domésticas/patologia , Síndrome , Proteínas Virais/análise , Proteínas Virais/imunologia , VirulênciaRESUMO
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.
Assuntos
Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Capsídeo/química , Capsídeo/genética , Proteínas do Capsídeo , Embrião de Galinha , Vírus da Doença Infecciosa da Bursa/química , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/diagnóstico , RNA Viral/química , RNA Viral/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
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.