Epidemic In Vestigation And Sequence Analysis Of Duck Hepatitis B Virus

In recent years,the harm of duck hepatitis B virus(DHBV)has been increasing in China.Due to the vertical transmission of duck hepatitis B virus,its infection in breeding ducks may cause greater damage to hatching and ducklings.In this study,a hybridization detection method with high specificity and sensitivity was developed for detection of DHBV,and epidemiological investigations were carried out in dead breed ducks,dead meat ducks,dead embryos and weak ducklings from different regions.Based on this,six DHBV wild strains were randomly selected for whole genome sequencing and analysis.This study will provide reference for the scientific prevention and control of DHBV infection in duck production.1.DHBV infection status in Shandong and Jiangsu provincesFive organs including heart,liver,spleen,lung and kidney were collected from 150dead ducks with clinical symptoms from 10 duck flocks in Shandong and Jiangsu provinces.Using the DNA extracted from tissue as a template,the infection status of DHBV was detected by PCR and the DHBV detection ratios of duck flocks and organs were counted.The results showed that the DHBV positive rate of flocks was 100%,and that of individual was41.3%.Among the tested organs,the DHBV positive rate of liver was 100%,and the DHBV positive rates of other organs were 77.4%in heart,71.0%in spleen,67.7%in lung and 66.1%in kidney,respectively.2.Development and application of DHBV probeA 423-bp highly conserved sequence in the DHBV C region,which was sequenced correctly after PCR amplification,was cloned and subsequently prepared into a digoxigenin labeled DNA probe.The result of specificity test showed that the prepared DNA probe was only positive for hybridization with DHBV DNA and negative for hybridization with other common pathogenic microorganisms.Sensitivity test results showed that the detection limit of the probe was about 3 pg DHBV DNA.The probe was used to detect 150 dead embryos,150sick ducklings,350 dead meat ducks and 360 dead breed ducks collected from 16 flocks in Shandong,Jiangsu and Anhui provinces.The results showed that the individual positive rates were 52%,50.7%,46.9%and 45.3%,respectively.At the same time,the 12 internal organs of the sick ducklings and the 5 organs of the adult ducks were detected.As a result,the positive rate of liver was the highest,reaching 100%.Comparing the detection results with the PCR results of the above samples,it was found that the positive rate of individual in the two detection methods had a coincidence of 100%,and the positive rate of various organs detected by the DNA probes was slightly lower than that of the PCR.The results showed that the prepared DIG-labeled DHBV DNA probe is highly specific and sensitive,and is suitable for large-scale diagnosis and epidemiological investigation of DHBV.3.The sequencing and analysis of DHBV wild virusSix DHBV wild strains from the most severely affected group of ducks randomly selected to amplify by PCR method and sequence,then homology analysis and phylogenetic tree were constructed with 15 DHBV reference genome sequences in GenBanks.The sequence analysis results indicated that the homology between the 6 genome sequences was89.8-99.8%,and the homology between the 6 wild strains and the other 15 reference sequences was 89.5-99.8%.Phylogenetic tree analysis showed that the 6 wild viruses were in the same branch as Chinese isolates.The amino acid homology of C protein between the 6wild viruses was 96.6-100%,and the homology between the 6 wild strains and the other 15reference strains was 96.2-100%;The amino acid homology of S protein between the 6 wild viruses was 89.7-99.7%,and the homology between the 6 wild strains and the other 15reference strains was 84.8-100%.The amino acid homology of P protein between the 6 wild viruses was 85.3-99.6%,and the homology between the 6 wild strains and the other 15reference strains was 84.8-99.5%.Combined with phylogenetic tree analysis,it can be found that C protein was highly conserved in the different DHBV strains,while S protein and P protein were more heterogeneous.4.Animal experimentA total of 100 3-day-old ducklings were injected with 200?L(6.6×10~5copies/?L)of DHBV-positive serum by intramuscular injection and these ducklings were placed in specific pathogen free(SPF)animal isolation system.Six ducklings were randomly selected every time to draw blood.Blood was collected at the 2ed day,the 3rd day,the 4th day,the 5th day,the 7th day,the 9th day and the 12th day,and sudsequntly blood were collected every other week.Viral blood volume was tested by DHBV SYBR Green I real-time fluorescence quantification PCR.The test results showed that the virus was detected on the 5th day in the blood,and the amount of virus reached the peak on the 12th day,and then gradually decreased,but there was no natural negative converse during the test period.The pathological changes of spleen and liver can be seen by necropsy.