| Duck viral hepatitis(DVH)is an acute and highly lethal infectious disease mainly caused by duck hepatitis A virus(DHAV),significant economic losses in duck farming industry.Two main viral genotypes,DHAV and DHAV-3 are prevalent in Chinese duck flocks.At present,there are some problems in the field of DVH prevention and control,such as the difficulty of vaccine strain cultivation with low antigen titer,and poor immune protection and antibody application in clinical practices.Therefore,it is urgent to elucidate the evolutionary characteristics of the current field isolates,clarify the molecular characteristics and variation of the epidemic strains,which definitely benefit the design of recombinant VP1 protein virus like particles,and explore efficient antigen preparation methods for new vaccine research.In this study,33 duck hepatitis clinical samples collected from Shandong,Jiangsu,Anhui,Sichuan,Hebei and Guangdong provinces since 2006 were isolated and identified,and one DHAV-1 and DHAV-3 isolates were selected for whole genome sequencing.Meanwhile combined with the complete gene sequences of DHAV-1 and DHAV-3 from Gen Bank database,the dataset was constructed respectively.The tempol signal was determined by Tree Time,and the best-fit model of DHAV-1 and DHAV-3 was determined by marginal likelihood estimation(MLE),and the Bayesian evolution analysis was conducted respectively.Maximum clade credibility(MCC)phylogenetic trees were inferred to compare the differences between the two genotypes.The vp1 gene of isolates was amplified and sequenced.Data-sets were constructed with the vp1 gene sequences of DHAV-1 and DHAV-3 from Gen Bank database,and the epidemiological trends of DHAV-1 and DHAV-3 were statistically analyzed.Spatial and temporal evolution and evolutionary rate differences were analyzed by BEAST v1.10,and vp1 evolutionary selection for DHAV-1 and DHAV-3 was analyzed by PAML.According to the analysis of genetic evolution,an epidemic isolate adapted to each genotype was selected as the immune antigen in the epidemic branch,and the recombinant VP1 protein virus like particles of DHAV-1 and DHAV-3 were constructed.The results as followed:1)A total of 26 isolates of DHAV were isolated from 33 suspected disease materials,including 9 isolates of DHAV-1 and 17 isolates of DHAV-3.Both DHAV-1 and DHAV-3datasets suggested tempol signals.The best-fit nucleotide substitution models,molecular clock models,and tree prior models for DHAV-1 models evaluated by MLE are TN+F+?4,Uncorrelated relaxed(Exponential),and GMRF Bayesian Skyride,respectively.The best-fit nucleotide substitution models,molecular clock models,and tree prior models of DHAV-3 models are TN+F+?4,Unrelated relaxed(lognormal),and GMRF Bayesian Skyride,respectively.The whole genome MCC tree indicated that DHAV-1 and DHAV-3 were of different evolutionary types,with great differences.They evolved independently.2)Statistical analysis of vp1 data set showed that DHAV-3 replaced DHAV-1 and became the dominant epidemic strain in 2012.Molecular evolution analysis of vp1 gene revealed the time to the most recent common ancestor and evolutionary rate of DHAV-1and DHAV-3.DHAV-1 is distributed in Clade 1.3.1 and Clade 1.3.2,and DHAV-3 is distributed in Clade 3.4.DHAV-1 strain(1910)evolved much earlier than DHAV-3 strain(1993).In addition,DHAV-3 underwent positive evolutionary selection,mainly adaptive evolution,while DHAV-1 underwent negative evolutionary selection.The evolutionary rate of DHAV-3 was significantly higher than that of DHAV-1,and the evolutionary rate of DHAV-1 was 4.890×10-4 95%HPD[3.072×10-4,6.830×10-4].The evolutionary rate of DHAV-3 was 2.125×10-3 95%HPD[1.686×10-3,2.567×10-3],which might be the main reason for the dominant epidemic strain.PAML identified vp1 selection sites for DHAV-1 and DHAV-3.For DHAV-1,amino acid sites 49(S),181(L),183(Q),184(G),187(*),193(N)and 219(Y)were positively selected,while for DHAV-3,amino acid sites were positively selected.There were positive selection effects at codon 49(S),185(Q),186(L),188(N),189(I),196(N)and 207(K).3)Using baculovirus expression system,DHAV-1 and DHAV-3 vp1 gene construct to expression vector p Fast Bac-Dual,through homologous recombination further formed the plasmid Bacmid-dvp1,after transfection 3?5 days,insect Sf9 cells produce obvious cytopathic effect,combined with WB and electron microscope,the results show that DHAV-1 and DHAV-3 recombinant VP1 protein formed self-assembled virus-like particles preliminarily.In conclusion,this study elucidated evolutionary characteristics of the current DHAV epidemic strains in the main duck breeding areas in China,and DHAV-1 and DHAV-3recombinant VP1 protein virus-like particles were constructed accordingly vaccine and provided scientific theoretical basis for prevention and control of DHAV. |
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