Genetic And Evolutionary Analysis Of DHBV In Anhui Province And Establishment Of A Duplex RT-qPCR Detection Method With DHAV-1

Duck hepatitis B virus(DHBV)was firstly identified in Peking duck sera in1980 and in the following years it was reported in Germany,South Africa,Australia,India and elsewhere,demonstrating the worldwide prevalence of DHBV.Currently,the prevalence of DHBV has been systematically reported in Sichuan,Hubei,Shanghai,Chongqing and Guangdong in China,but little is known about DHBV epidemiological and evolutionary status in Anhui Province.In order to understand the natural infection and structural characteristics of the genes of hepatitis B virus in ducks in Anhui,120 duck liver tissue samples were collected from Hefei,Anqing and Bengbu in Anhui for testing.28 DHBV were confirmed postive for DHBV using specific primers,and the complete genome sequences of 5 of the DHBV-positive samples were amplified after verifying their accuracy,cloned and sequenced,and genetic evolutionary analysis and Bioinformatics analysis were performed based on these sequences.The results showed that the nucleotide similarity of the five strains was 94.5～96.3%and 93.2～97.9%with other reference strains in Gen Bank.The phylogenetic tree analysis showed that all five DHBV strains belonged to the"Chinese"isolates.Secondly,three potential intraspecific recombination events were predicted using the RDP5.0 software,mostly in the ORF-Pre C/C region.Biological prediction of the DHBV C protein showed that it is a hydrophilic stability protein with no signal peptide and no transmembrane region,with a total of 44 phosphorylation sites.On selection pressure analysis,two positive selection sites on the C protein were found to overlap with B-cell epitopes.In this study,specific primers were designed for the conserved regions of DHBV ORF-Pre C/C and DHAV-1 VP0 genes,and a duplex SYBR Green I-based real time fluorescence quantitative PCR(RT-q PCR)assay was established by optimizing the reaction conditions and system,with the following results.In the sensitivity assay,the minimum detection level was 8.5×10~1copies/μL for DHBV and 2.8×10~1copies/μL for DHAV-1,both of which were 100 times higher than the minimum detection level of normal PCR.No cross-reactivity with other duck viruses was observed in the specificity assay.The methods showed good reproducibility with coefficients of variation all below 5%and high reliability.The q PCR method is also significantly better than the common PCR method for clinical sample detection.The method has the advantages of rapidity,reliability,sensitivity and specificity.In summary,this study successfully amplified five strains of DHBV in Anhui and performed genetic evolutionary analysis,and established a duplex SYBR green I-based RT-q PCR assay.This study enriches the genetic evolution data of DHBV and further understanding the genetic characteristics and molecular features of DHBV,and providing information on the genetic evolution of DHBV.It also provides a useful tool for clinical diagnosis and epidemiological investigation of DHBV and DHAV-1.