RESUMO
Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.
Assuntos
Vírus da Anemia da Galinha/patogenicidade , Animais , Sequência de Bases , Proteínas do Capsídeo/química , Vírus da Anemia da Galinha/classificação , Vírus da Anemia da Galinha/genética , Galinhas , Dados de Sequência Molecular , FilogeniaRESUMO
A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.
Assuntos
Vírus da Anemia da Galinha/genética , Vírus da Anemia da Galinha/isolamento & purificação , Galinhas/virologia , Animais , Antígenos Virais/metabolismo , Medula Óssea/virologia , Linhagem Celular , Vírus da Anemia da Galinha/metabolismo , Clorofórmio/farmacologia , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Ágar , Técnica Indireta de Fluorescência para Anticorpo , Temperatura Alta , Malásia , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Pele/virologia , Temperatura , Timo/virologia , Fatores de TempoRESUMO
Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
