RESUMO
Abstract:Subgroup J avian leukosis virus (ALV-J) infect cells by binding to the chNHE1 receptor protein of the host and causes tumors. The tumor incidence of the ALV-J-infected chickens was observed by histo pathology, and virus was isolated on DF-1 cell line. The ALV-J load and mRNA of chNHElreceptor protein were detected by real time PCR. The relationship between ALV-J load, chNHE1 receptor expression levels and tumor spectrum was analyzed. The results showed that the tumors induced by ALV-J in laying hens and local lines of chicken were different. No significant relationship was observed between ALV-J load and tumor spectrum. ALV-J load was positively correlated with mRNA expression of chNHE1. The mRNA expression of chNHE1 increased when the tumors occurred. Our results suggest the chNHE1 protein is not only the receptor of ALV-J infected host but also play an important role in the process of tumor development. This study provides a scientific basis for further studying of oncogenic mechanism of ALV-J.
Assuntos
Animais , Leucose Aviária , Genética , Metabolismo , Virologia , Vírus da Leucose Aviária , Genética , Fisiologia , Galinhas , Genética , Metabolismo , Doenças das Aves Domésticas , Genética , Metabolismo , Virologia , Receptores Virais , Genética , Metabolismo , Trocadores de Sódio-Hidrogênio , Genética , Metabolismo , Carga ViralRESUMO
The transmembrane protein (TM) encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs. Several highly conserved regions in TM are important targets for antivirus studies. Studies on structure and function of TM will provide basic information for anti-retrovirus, especially for avian leukosis virus. In the study, gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701. The gp37 gene was cloned into pMD18-T vector, and was sequenced. Then, pFast-BacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system. The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot. It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.
Assuntos
Animais , Leucose Aviária , Virologia , Vírus da Leucose Aviária , Classificação , Genética , Linhagem Celular , Galinhas , Clonagem Molecular , Expressão Gênica , Spodoptera , Proteínas do Envelope Viral , Genética , MetabolismoRESUMO
During July to November in 2007, several outbreaks of Hemangiomas in Hy-line Brown laying hens were observed in China. The virus that infected these flocks was identified in cultured DF-1 cells by PCR and indirect fluorescent assay (IFA) with ALV-J specific monoclonal antibody JE-9. The gp85 gene of one strain named WS0705 of ALV-J was cloned and expressed. Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain had the highest homology with that of the prototype HPRS-103. The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector. rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system. The rBac-WS0705gp85 protein was analyzed by indirect immunofluor escence assay and Western blot. The results showed that positive green fluorescent was present in Sf9 cells infected with the recombinant virus and a 35 kD band was present in western blot. It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with the recombinant virus and the SU protein of WS0705 can bind specifically to JE9 MAb of ALV-J. The expressed protein can be used to detect hemangiomas induced by ALV-J.
Assuntos
Animais , Sequência de Aminoácidos , Vírus da Leucose Aviária , Classificação , Genética , Western Blotting , Linhagem Celular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Hemangioma , Virologia , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Proteínas do Envelope Viral , Química , Genética , MetabolismoRESUMO
Two strains of Avian leukosis virus subgroup B (ALV-B) were isolated for the first time in China Hy-line White on the cultured DF-1 cells which were inoculated tissue samples from by an ELISA assay, a histopathology examination and a PCR-based diagnosis. The results from the ELISA assay indicated that the positive rate of serum antibodies to ALV-B and ALV-J virus were 16.3% (15/92) and 13% (12/92), respectively. The histopathological examination indicated that two types of tumor cells existed at same focus in liver and spleen, which mainly were myelocytoma cells and lymphosarcoma cells. The PCR-based diagnosis were performed as follows: the cellular DNA was extracted from the inoculated DF-1 cells; the specific fragments of 1100 bp and 924 bp were obtained by a PCR system with the diagnostic primers of ALV-B and ALV-J; and the PCR results for ALV-A, MDV and REV were all negative. Then, the amplified fragments of the two ALV-B stains were partially sequenced and shown an identity of 92.8%,94.7% with the prototype strain of ALV-B (RSV Schmidt-ruppin B). The identities of two ALV-J strains with the prototype strain HPRS-103 at 96.9%, 91.5%; The identities of two ALV-J strains with the American prototype strain at 85.9%, 81.5%. Our study had shown that ALV-B was isolated for the first time from the ALV-J infected commercial layer flocks in China. It also indicated that the chance of genetic recombination among various subgroups of ALV was increased.