Screening Attenuated Vaccine Strain Of Duck Hepatitis A Virus Serotype 3

Duck Hepatitis A Virus（DHAV）is the causative agent of duck viral hepatitis（DVH）.DHAV have three serotypes as DHAV-1,DHAV-2 and DHAV-3,and there is no cross immune protection among them.In recent years,cases of ducklings infected with DHAV-3 have occurred in many places in China,the harm is serious.It is urgent to study safe and effective DHAV-3 vaccines.In this study,We obtained a DHAV-3 attenuated strain by continuous passage of chicken embryo yolk sac inoculation,which was safe,stable virulence and good immunogenicity to ducklings,and could provide virus materials for further development of attenuated vaccine.The main contents of this study are as follows:1 Cultivation of attenuated strainA duck hepatitis a virus serotype 3 strain was obtained by continuous passage of egg yolk sac inoculation.The median lethal dose(ELD₅₀)of 90th generation virus（DHAV-3LCF90）and 100th generation virus（DHAV-3 LCF100）on chicken embryos were10^-7.44/0.3m L and 10^-7.80/0.3m L,respectively.By RT-PCR or PCR,the strain could only detect nucleotide fragment of DHAV-3,while DHAV-1,DHAV-2,duck statin virus,duck reovirus,duck tambusu virus,duck fever,avian influenza and avian adenovirus were negative.2 Sequencing of the whole genome of virusThe whole genome nucleotide sequence of DHAV-3 LCF90 virus was 7777nt,including5’UTR（651nt）,1ORF（6756nt）,3’UTR（336nt）and poly（A）tail.The homology and phylogenetic tree analysis of DHAV-3 LCF90 virus and other 24 strains of DHAV-3 virus showed that the virus had the highest similarity with NC strain（KU860089.1）,the similarity of whole genome nucleotide sequence and polyprotein amino acid sequence was 98.9%and97.7%,respectively.The whole genome sequence between the DHV-3 LCF90 virus and virulent strain DHAV-3 LC virus showed no base insertion or deletion,but there were 32 base mutations.There were 6 base mutations were located in the noncoding region and 26 in the ORF region.In ORF,9 base mutations resulted in amino acid changes,of which the structural protein had only one amino acid mutation at the C-terminus of VP0 protein(N₂₅₄→D₂₅₄).The nucleotides and coding amino acids of the VP0 gene of different generations of viruses were gradually mutated during the adaptation of DHAV-3 LC virus to chicken embryos.The nucleotide of the DHAV-3 LC virus was C₁₄₁₀A₁₄₁₁(amino acid residue was N₂₅₄),the nucleotides of DHAV-3LCF20 to DHAV-3 LCF50 viruses were C₁₄₁₀G₁₄₁₁(amino acid residue was D₂₅₄),and the nucleotides of DHAV-3 LCF60 to DHAV-3 LCF100 viruses were T₁₄₁₀G₁₄₁₁(amino acid residue was D₂₅₄).3 Pathogenicity to ducklingsThe 1-day-old ducklings were subcutaneously inoculated with 10^8.0ELD₅₀/0.5m L of DHAV-3 LCF90 in the neck,and all of them survived,normal growth and no clinical manifestations during the 7 days observation period.The serum alanine aminotransferase（ALT）at 72h and 168h after duckling inoculation was not statistically different from the healthy control group（P>0.05,n=5）,and was significantly different from the DHAV-3 LC virulent infection group（P<0.01,n=5）.There were no visual pathological changes and no obvious structural abnormalities in the culling ducklings,while the ducklings in the virulent infection group showed gross and histopathological changes of typical duck hepatitis a virus infection.4 Virulence stability to ducklings10^8.0ELD₅₀/0.5m L DHAV-3 LCF90 was subcutaneously inoculated with 1-day-old ducklings through the neck.At 72h culled the ducklings after inoculation and collected the liver tissue to prepare homogenate fluid（1:5）.The homogenate fluid was inoculated again with ducklings and passed through continuously for 5 times.During inoculation and passage,the survival,growth and autopsy observation of ducklings were normal.There was no statistical difference between serum alanine aminotransferase and the blank control group（P>0.05,n=5）.Liver tissue had no obvious pathological histological changes,but DHAV-3nucleotide fragment could be detected from liver tissue by RT-PCR,and DHAV-3 could be isolated from liver tissue homogenate（1:5）by chicken embryo inoculation.Chicken embryos were inoculated with liver tissue homogenate（1:5）from the 1st generation to the 5th generation,and the ELD₅₀of chicken embryos were respectively 10^-4.16/0.3m L,10^-4.25/0.3m L,10^-4.57/0.3m L,10^-5.12/0.3m L and 10^-4.22/0.3m L.5 Determination of protection and minimum immune dose1-day-old ducklings were subcutaneously immunized with 2×10^5.8ELD₅₀/0.3m L of the DHAV-3 LCF100 in the neck,10^2.25LD₅₀/0.2ml of DHAV-3 LC strain was injected into the neck of ducklings to challenge after 3 days.The results showed that 100%of ducklings survived in the immunized group（10/10）,and 10%（1/10）in the blank control group.1-day-old ducklings were subcutaneously inoculated in the neck with different doses of DHAV-3 LCF100,and the challenge protection index of attack was determined after 3 days.The results of three repeated tests showed that the protection index of attack was above 85%after the 1-day-old ducklings were inoculated with 2.5×10^4.8ELD₅₀/0.3m L and above doses of virus.The above research results indicate that a DHAV-3 attenuated strain was obtained by continuous passage of chicken embryo yolk sac inoculation,which was not pathogenic and stable in virulence to ducklings,and could induce good immune response in ducklings.