RESUMO
To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8％-99.7％ similarity at the nucleotide level and 92.4％-99.6％ at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.
RESUMO
To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China,the complete genomes of six DuCV strains,which were detected from Cherry Valley ducks in China between 2007 and 2008,were sequenced.Sequence and phylogenetic analysis were carried out to compare these six strains with another 27DuCV strains from Mulard duck,Muscovy duck,Pekin ducks and Mule duck.The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV,such as a stem-loop structure,three major open reading frames (Rep,Cap and ORF3),four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein.Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes.The DuCV strains with complete genomes containing 1988and 1989 nucleotides clustered in genotype A,whereas the strains with complete genomes containing 1991,1992,1995 and 1996 nucleotides lay in genotype B.The six DuCV strains from Cherry Valley ducks were divided into the two groups.The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.
RESUMO
The duck circovirus (DuCV) infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province.Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%～97.5% nucleotide sequence identity to all the four Taiwan isolates (TC1/2002, TC2/2002, TC3/2002, TC4/2002), 82.9% identity to the America (33753-52) isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however,showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.
RESUMO
There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene and 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP and pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF) and pp38-deleted derivative rMd5deltapp38 infected CEF (rMd5deltapp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF, and no EGFP could be detected in uninfected CEF or rMd5deltapp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5deltapp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5deltapp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24 and EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38 and pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.