| Duck viral hepatitis (DVH) is an acute, highly contagious, viral disease of young ducklings. The main characterization of DVH is hepatitis. The agent of DVH is duck hepatitis virus (DHV). Three distinct serotypes have been detected, termed as DHV-1, DHV-2 and DHV-3. No cross-reactive are detected among the three serotypes. DHV-1 is the most important and severe one. It is of economic importance in all duck-raising areas of the world.Sixteen DHV-1 viruses were isolated from outbreaks in ducks in China between 2003 and 2010. All the viruses were isolated from liversof the suspected ducks using specified pathogen-free (SPF) duck embryonated eggs. At least three blind passages were performed until the characteristic embryo changes were observed within 72h post inoculation. Gerenrally, typical signs including heamorrhage and death of embryo were observed in the first or second passages inoculated by DHV-1. Genomes of the 16 strain were determined by RT-PCR and 3`RACE. The phylogenetic tree based on the 16 genomes and other picornaviruses showed that DHV-1 had a far relationship with viruses of different genuses in family Picornaviridae, indicating that DHV-1 should be a new genus in family Picornaviridae. Phylogenetic tree based on the 16 isolated strains and other 64 reference strain indicated that DHV-1 could be clustered into three different genotypes, named genotype A, B and C. The main genotype in China was genotype A. Members in genotype C were also isolated in China. The alignment of the genomes indicated insertions in VP1 gene and 3`-end of genomes of genotype C compared with members in genotype A. The identity of members in the same genotype was over 90%, and the identity of viruse in different genotypes was less than 80.0%.Du/CH/LBJ/090809 was selected as presentative of genotype A for the pathogenicity. The Du/CH/LBJ/090809 could lead to 30% mortality of 11-day-old specific pathogen free (SPF) ducklings in the challenge test, implicating that the virus had high virulence to ducklings. Seven different of recombinant proteins of Du/CH/LBJ/090809 were expressed to establish ELISA to evaluate the antibody post inoculation of Du/CH/LBJ/090809. The result showed that the recombinant proteins VP0 and VP1-2 could be used as antigens in ELISA. The antibody could be detected 8 d post inoculation (dpi) of Du/CH/LBJ/090809. At 12 dpi the antibody reached the peak. Antibody did not disappear until 32 dpi.Then the Du/CH/LBJ/090809 was used to passage serially in SPF chicken embryonated eggs. Viruses of passage levels 29 (P29) and 40 (P40) was used to evaluate the attenuation to duckling by inoculating the two levels of viruses to 11-day-old SPF ducklings. The result showed that both the P29 and P40 were fully attenuated and were non-pathogenic to the duckling. Additionally, the inactivated P40 was used as vaccine in vaccination-challenge test and the result indicated that the inactivated virus P40 could afford protection against the pathogenic parent virus by clinical response. Furthermore, the nucleotide and amino acid sequences of the complete genomes of parent Du/CH/LBJ/090809, P16, P29 and P40 viruses were compared. Nucleotide and amino acid changes, which might be accounted to the decrease in virulence by embryo-passage, were observed among the genomes of parent Du/CH/LBJ/090809, P16, P29 and P40 viruses.A full length cDNA clone of Du/CH/LBJ/090809 P40 was assembled in the plasmid vector pACYC-177. RNA synthesized in vitro by using T7 promoter in front of the cDNA produced infectious viral particles after transfection of BHK cells. The lysates were inoculated into SPF chicken eggs until the appearance of pathological changes. The generation of the infectious cDNA of DHV-1 will provide useful tools for understanding the evolution, pathogenesis and functions of viral genes. |
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