Effects Of Amino Acid Mutation At Position 38 And 144 Of 3C Gene On The Proliferation And Partial Bioactivity Of Duck Hepatitis A Virus Type 1

Duck hepatitis A virus(DHAV),belonging to the Avihepatovirus of Picornaviridae,can cause duck viral hepatitis(DVH),which is a short duration,highly fetal infectious disease.DVH mainly threatens the ducklings within one month of age and brings huge economic losses in duck industry.3C protease is one of the important proteases of Picornaviridae virus,which plays an irreplaceable role in the replication process of virus.In this study,to determine the effects of amino acids mutations on viral proliferation capacity and partial biological activity,we mutated the amino acid at the position of 38 and 144 in 3C gene by PCR point mutation based on the DNA-Launched infectious clone of DHAV type 1(DHAV-1).1.Prokaryotic expression and preparation of antiserum of 3C proteinThe anti-3C serum was produced to carry out the IFA identification of DHAV-1 infectious clones in this study.Firstly,two amino acids sited in 38 and 114 were mutated alone and simultaneously in the wildly 3C gene,which was named 3C38,3C144 and 3C38+144 respectively.Prokaryotic expressions were performed by cloning the three mutations and the wildly 3C gene into vector pGEX6P-1.The positive plasmid was transformed into Escherichia coli BL21(DE3)to induce fusion protein induced by IPTG.Purified fusion protein was mixed with complete adjuvant of Freund's to immunize BALB/C mice,each mice were inject 100 g.Second,third and strengthen immunization were performed using same quantity protein that mixed with incomplete adjuvant of Freund's every two weeks.After seven days of strengthen immunization,antisera were taken from the blood of mice by centrifugation.The result of ELISA showed that the serum titer of 3C38 was much higher than these of the other three groups.2.Construction of 3C-mutation-gene infectious cloneThe,using the Designing primers based on the sequence and restriction map of DHAV-1 infectious clones stored in the laboratory to amplify 3C38,3C144,3C38+144 and 2C3 A.Fusing these fragments by PCR and constructed positive plasmid of 2C3A3C38,2C3A3C144 and 2C3A3C38+144 respectively.Sequencing results showed that those mutations shared 100% homology with the 3C sequence in the parent virus except artificial mutations.The mutated 3C gene was assembled to the whole genome referenced to the restriction map of DHAV-1 infectious clones.The 2C3A3C38,2C3A3C144 and 2C3A3C38+144 fragments were fused with 3D fragment to obtain 2C3A3C3D(hereinafter abbreviated CD)fragments with the mutations,which was assembled into the whole genome,using the EcoRV restriction site existing in the whole genome.Sequencing results showed that the mutations were found in38 th amino acid and 144 th amino acid in the site-directed mutagenesis of 3C gene and the rest of the amino acids were not mutated,meaning the positive recombinants SR38,SR144 and SR38+144 were obtained.The recombinant plasmids SR38,SR144,SR38+144 were transfected into BHK-21 cells,at the same time,parental SR plasmid was used as positive control while sterile PBS was used as negative control.We didn't find any obvious CPE in BHK-21 cells after transfection while fluorescence quantitative PCR and IFA test showed positive.Gene sequencing indicated that the virus was rescued successfully.3.Detection of virus proliferation and the partial biological activity of the rescued virusBHK-21 cells were re-infected with SR38,SR144,SR38+144 groups and the female virus,collected after transfection of BHK-21 cells for 48 h.The growth trend of each saved virus was made according to the number of virus copies,measured at 24 h,36h,48 h,60h and 72 h.The growth trend of each rescue virus showed that both the proliferation cycle and the maximum copy number of the rescue virus that with different mutations in the 3C gene was different from the female virus,indicating that the mutation of the 38 th amino acid and the 144 th amino acid in the 3C gene had caused a certain impact on the proliferation of the virus.A total of 65 healthy duck embryos(9-day-old)were divided into five groups to study the biological diversity of the virus.A 300 ?L sterile PBS was injected by allantoic cavity approach in the first group,Rescue virus of SR38,SR144,SR38+144 and the female virus SR were injected by same method respectively.Allantoic fluid was collected at the time of 24 h,48h,72 h,96h and 120 h to detect viral proliferation by QRT-PCR,at the same time,the clinical symptoms of duck embryos in different groups were observed.The result indicated that there were significant differences both in the proliferative cycle and virus c opy number between the saved virus and the female virus.Meanwhile,bleeding spots were clearly found in liver,brain and skin of embryos.