Characterization Of Bovine Adenovirus 3 Isolated In China And Complete Genome Analysis

Bovine adenovirus type 3(BAV3)belongs to the Mastadenovirus genus within the familyAdenoviridae and is the representatives of subgroup 1 which grow relatively well in established bovinecell lines and contain a common complement-fixing antigen. BAV3 is considered one of the importantrespiratory tract pathogens of cattle, particularly newborn calves. The major clinical symptoms of BAV3infection is pneumonia and other respiratory disease which often associated with mixed infection andsecondary infection. BAV3 also can cause newborn hamster tumors. Recently, calf pneumonia is acommon disease in China which causes huge economic losses in cattle industry. This disease is popularin the world since BAV3 was first isolated by Darbyshire and his co-worker in 1965 from theconjunctiva of an apparently healthy cow. At present, there is no report on BAV3 infection and isolation,and no related diagnostic reagents and vaccine in China.A virus was isolated from affected cattle which showed intense respiratory clinical signs includingnasal discharge and labored breathing with body temperature above 40℃in 2009. The supernatant ofnasal swabs after treated was inoculated on MDBK cells and CPE characterized by cytoplasmic bridgewas detected microscopically after two blind passages. The virion was observed by electron microscopywhich was symmetric three-dimensional structure and about 70nm size. Direct immunofluorescence testshowed that the reaction between anti-BAV3 polyclonal antiserm conjugated to fluoresceinisothiocyanate and the isloate was positive. HA test showed that the isolate could agglutinate bovineerythrocytes with HA titer of 1:64, but for the red blood cells of mice, rats, guinea pigs, chickens, horsesand sheep without aggregation. Heating sensitive assay showed that the TCID50 of the isolate could bedropped 2.2 titer at 50℃15min, and could completely inactivated at 56℃30min, which consistentwith the subgroup 1 of bovine adenovirus. According to the published complete genome sequence ofBAV3, a special pair of primers was designed on E2A gene. With the DNA template extracted from theisolate, the 644bp gene fragment was amplified by PCR. And then, the PCR product was cloned andsequenced. The phylogenetic tree analysis based on E2A gene showed that the isolate closely related toBAV3. The alignment of DBP encoded by E2A gene showed that the isolate has the highest homologywith the reported BAV3, above 98%. The above results showed that this isolate was BAV3, anddesignated as BAV3 HLJ0955. It is the first time that BAV3 has been isolated in China.According to the published complete genome sequences of strain WBR-1 (GenBank accession no.AF030154) , 42 pairs of primers was designed, and the complete genome sequencing of HLJ0955isolate showed that the genome is 34,132 bp in length with a G+C content of 53.6% and A+T content of46.41%. Compared with strain WBR-1, we found that HLJ0955 isolate contained some successivedeletions in region ITRs, E1B and E4 ORF1. But Some gene regions of HLJ0955 isolate were similar tostrain WBR-1, with 92.7%-100% nucleotide and 87.2%-99.1% amino acid similarity. The phylogenetic tree analysis based on the complete genome of adenoviruses showed that HLJ0955 isolate correspondswell to strain WBR-1, which further confirmed that HLJ0955 isolated in China belonged to BAV3.In this study, we identified and sequenced BAV3 HLJ0955 isolated in China for the first time, andanalysed its biological characterization and the molecular structure. The result would contribute to thedevelopment of new vaccines and diagnosis techniques.