| In recent years,duck adenovirus infections characterized by hemorrhagic hepatitis have spread in duck farms in China.It was diagnosed as duck adenovirus type 3（DAd V-3）infection with the morbidity rate is 40%to 55%,causing heavy losses to the duck breeding industry.In this study,the liver of a duck with suspected pericardial effusion-hepatitis infection was collected for virus isolation and identification.As a result,one strain of DAd V-3 was successfully isolated.After purification,the pathogenicity studies and viral genome sequencing were carried out.Through prokaryotic expression of DAd V-3 Fiber-2 protein,monoclonal antibodies against Fiber-2 was prepared.1.To isolate DAd V-3 in clinic,the livers of dead ducks were inoculated into the monolayer of chicken hepatocellular carcinoma cell line（LMH）,and the genomic DNA was extracted from the cell supernatant.After PCR identification and sequencing analysis,the virus was identified as DAd V-3 and designated as strain TZ193（Gen Bank:MT934842）.2.In order to identify the molecular difference between the isolate and the classic strain of DAd V-3,the full-length of viral genome of the isolate TZ193 was sequenced.The result showed that its total length was 43842 bp,and G+C content was 47.16%.The phylogenetic tree of the genome sequence showed that the TZ193 strain and the DAd V-3 CH-GD-12-2014 strain is in the same branch.Compared with the CH-GD-12-2014 strain,the TZ193 strain had a point mutation in the ORF67 gene,which causes the gene to terminate prematurely,indicating that DAd V-3 virus has been mutated.3.In order to quickly and accurately detect virus loading when DAd V-3 infection, Taq Man probe quantitative polymerase chain reaction（q PCR）method was established.The result showed that the linear equation was y=-3.3895x+42.668,R2=0.9958.The concentration limit of the plasmid that can be detected is 45.96 copies/μl.The coefficient of variation is 0.29%～1.20%.The specificity and cross-reaction assay were also detected.4.After the virus was purified,animal experiment was carried out.Ten 1-day-old ducks were intramuscularly inoculated with 1.58×106TCID50/m L.It was found that all the ducklings died after 4 days of inoculation,and the lethality rate could reach 100%.At necropsy,liver hemorrhage and pericardial effusion were observed.The pathological changes,such as degeneration of cardiac muscle fibers and necrosis of liver cells,were observed in the tissue section.Real-time quantitative PCR method showed that virus loading in all tissues and organs.Among them,the level of virus in cecal sample was the highest by reaching 8.4×105 copies/μL,followed by the duodenum and liver and heart,however,that in the brain was lowest.5.To obtain specific antibodies against DAd V-3,fiber-2 gene was selected as interest antigen to prepare monoclonal antibodies（m Abs）.The fiber-2 gene was amplified by PCR and subcloned into p ET32a prokaryotic expression plasmid.The recombinant plasmid was transformed into BL21（DE3）and induced by IPTG.By SDS-PAGE identification,the bacteria were collected and the protein was purified to immune mice for three times.The monoclonal antibody was prepared using hybridoma technology,and the positive hybridoma cells were screened and develop subcloning culture.The obtained m Abs were identified by indirect immunofluorescence assay（IFA）and specific fluorescence was found in the cells.Western blot identification revealed that a specific band appeared at about 59k Da.In conclusion,one strain of DAd V-3 was isolated and identified in this study.The full-length of viral genome was sequenced.Taq Man probe quantitative polymerase chain reaction method for DAd V-3 was established.After the virus was purified,animal experiment was carried out.The monoclonal antibody against Fiber-2 was prepared.This study provide a basis for an in-depth understanding of the evolutionary process and pathogenic mechanism of DAd V-3 and antigen selection for the preparation of DAd V-3 subunit vaccines. |
