| Chicken anaemia virus (CAV) is a virus causing anaemia accompanied by aplasia of the bone marrow and atrophy of the lymphoid organs in young chickens and a transient severe immunodeficiency in older chickens. In this study ,five Chinese CAV isolates were detected positively using dot-blot hybridization with CAV genomic DNA probe. The VP3 genes (0.35kb) of the five Chinese isolates were amplified and sequenced . The VP3 sequences were all identical to the VP3 sequence of CIA-I strain (U.S.A.), there was only 1 nucleotide different from CUX-I strain (Gremany ) (Cæ£~T at 674nt) , but same to TR2O strain (Japan) and CAU269-7 strain (Austrailia) .The sequence differences in VP3 gene were restricted to the 5?terminat (158bp) region, the 3?terminal sequences were conserved in different CAV isolates. VP3 was a protein of 121 animo acids containing two proline-rich stretches and two Arginine-rich basic regions. The changed nucleotides were not affect those important functional regions.Three fragments (1.Okb,0.8kb,0.6kb) which were included full length genomes were amplified by polymerase chain reaction (PCR) from the genomic DNA of two Chinese isolates ,HA9805 strain and JN0007 strain. The 1.5kb sequence (including 1.0kb and 0.6kb fragments ) wered compared between the different CAV strains. Two Chinese isolatesHA9805 and JN0007, demonstrated overall nucleotide sequence homologous to each of CUX-1,CIA-1,CAU269-7,and TR2O strains (more than 95%). A phylogenetic analysis results indicated that CAU269-7 wasLf~phylogenetically distinct from all other isolates ,HA9805 and JN0007 appeared to be most closely related to CUX-l and TR2O respectively. The sequence coding C-terminal of VP2 (also including VP3 gene) was very conserved. The variations of nucleotides were located on other regions randomly . The most nucleotide variations were synonymous. However the biological characterization of the non-synonymous nucleotide changes was still not clear. The noncoding region of genome of HA9805 and JN0007, also contained motifs considered to be involved in DNA replication ,translation and transcriptional activity which were reported in other CAV strainsA recombinant plasmid (pCAV 2.4,5.0kb) which contained a incomplete EcoRI site (-GACTTC-) in the non-coding region and couldn抰 be re-circlized. In order to obtain infectious clone, CAV genomic DNA was amplified by PCR using the pCAV2.4 plasmid DNA as template and cloned into pUC18 vector (pCAVE~,5.0kb). The incomplete EcoRL site (-GACTTC-) in pCAV2.4 was point-mutated into the complete EcoRI site (-GAATTC-) in pCAVE~. The 2.3kb CAV genomic DNA was extracted from the recombinant pCAVE~ digested with EcoRI, then re-ligated, and transfected the host cell line (MSBI). After 8 passages ,the cell culture medium didn抰 turn yellow. The supernatant was inoculated into 1 day old SPF chickens . Typical anaemia and thymus atrophy were shown after 14-18 days post infection. However the circular pCAVE~ plasmid DNA, which contained single copy of CAV RF genomic DNA ,couldn抰 produce reproductive virus after being directly transfected MSB1 cells.The 2.3kb CAV genomic DNA extracted from pCAVE~, when was inserted into the pCAV2.4 at EcoRJ site, a plasmid pCAV2E + (7.3kb ) was constructed, that contained cloned tandemly-repeated CAV genomic DNA. Infectious virus was recovered from MSB1 cells transfected with the circular pCAV2E~ plasmid DNA. These results indicated that the re-ligated genomic DNA (2.3kb) and the tandemly-repeated CAV RF fragments were all capable to produce infectious virus following transfection into MSB1 cells. These results will be very useful in the further study on construction low or non-pathogenicity virus by mutation on genome.Expression of three virus-encoding proteins in vitro were studied. The whole length VP1 gene and the large fragment of VPI without N-terminal 21 amino acids and C-terminal 4 amino acids ,which were inserted into pGEX-5X-3 vector following GST gene in correct orientation, were not expressed in E coli. by...
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