Molecular Epidemiological Analysis Of Duck Hepatitis B Virus In Cherry Valley Duck In Henan Province And Anti-duck Hepatitis B Virus Activities Of FNC In Vivo

Hepatitis B is one of the major diseases which endanger human health. There are about 400 million hepatitis B patients or carriers in the world. But so far, vaccine and chemical drugs are most commonly used for the prevention and cure of hepatitis B. In China, the drugs for the treatment of hepatitis B are mainly relying on imports or imitation. There is no original drug with significant clinical effect and independent intellectual property rights. The natural infection rate and molecular epidemiologic characteristics of DHBV isolated from cherry Valley Duck in Henan province were investigated. With the SYBR Green I Real-time PCR assay for quantification of DHBV DNA and pathological detection, the model of cherry Valley Duck infected by DHBV was established and used to evaluate the antiviral effect of FNC (a new drug for hepatitis B). The results were as follows.In order to clarify the natural infection rate and the genome structure of DHBV in Cherry Valley Duck of Henan province. The serum of Cherry Valley Duck was detected by PCR from Xinyang, Xinzheng and Jiaozuo in Henan province. The complete genome of the DHBV strains isolated from each area were amplified by PCR and cloned into T vector and sequenced. The results showed that the natural infection rate of DHBV was 10.5%,8.9%,42.9% in Xinyang,Xinzheng,Jiaozuo respectively. These three isolated DHBV complete genomes had 3021,3027,3024 nucleotides with three overlapping open reading frames encoding the surface(S),core(C) and the polymerase(P) proteins, and the sequence homology was from 89.3% to 93.5%。The amino acid sequences of the S protein and C protein were highly reserved,but the P protein were significant variation. Comparison of genome sequences of these strains with those of 19 DHBV strains from the GenBank revealed an identity from 89.4%%to 99.5% at the nucleotide level. This finding was corroborated by a phylogenetic tree analysis. Therefore,the DHBV Xinyang strain should be classified as a subtype of the Western strains, and the other two strains should be classified as a subtype of the Chinese strains. This study further shows that there is a distinct signal peptide in the 1～35aa area, a breakpoint in the 27～29aa area of S-ORF, and antigenic sites are mainly distributed in the amino acid sequencing of PreS area. Finally the DHBV Natural infection from Henan Cherry Valley Duck is mastered. The successful clone and analysis of DHBV provides proper theory foundation and useful practice value for further study.A rapid and specific SYBR GreenⅠReal-time PCR assay was established to detect DHBV DNA. A pair of primer was designed based on the sequences from Günther, and used for amplifying the genome of DHBV DNA,the amplified PCR fragments were cloned into pMD-18-T as standard plasmid. Based on the conserved sequences of DHBV S gene, another pair of primer for Real-time PCR was designed and used to amplify the standard plasmid for constructing the standard curves. Meanwhile, the specificity, sensitivity and repeatability of the assay were tested. The results showed that the Real-time PCR assay was highly specific and had a broad linear detecting range (7.02×108 ~ 7.02×104copies /μL, R=1.00) with 105% PCR amplification efficiency. It had a detection threshold of 70 copies of plasmid DNA and was high sensitive. The established SYBR GreenⅠReal-time PCR for detecting Duck hepatitis B virus is rapid, specific and sensitive, and can be used a useful tool for evaluating effect of anti-HBV drug.To study the inhibitory effect of the extract from FNC on DHBV in vivo and to provide experimental evidence for studying and developing new anti-HBV drugs. Three days-old Henan Cherry Valley ducklings infected with DHBV were used as the hepatitis B animal model.Positive ducklings were detected by PCR 7 days after infection,and randomly divided into five groups (16 ducks per group):FNC high-dose group,middle-dose group,low-dose group,Lamivudine positive control group and negative control group.Every group was i.g. for ten days.The quantity of DHBV-DNA in serum and liver was monitored by Real-time PCR. ALT and AST activity in serum were detected by Automatic biochemical detector, and liver histopathological examination by hematoxylin and eosin stain at T0 (1 day before treatment ),T5 (the fifth day after drug usage),T10 (the tenth day after drug usage) and P3 ( the third day after drug withdrawal ). At 5 days and 10 days after oral administration with FNC,the loads of DHBV-DNA from the blood and hepatic tissue were obviously lower than that of negative control group , there was significant difference(P<0. 05) between the two groups.DHBV-DNA level in serum and hepatic tissue return trivial at the third days after drug withdrawal.Compared with FNC high-dose group and Lamivudine group,there was no obvious difference(P>0.05).Compared with the normal saline control group, there was significant difference in the level of ALT, AST in FNC groups on the earlier stage of treatment, but no significant difference on the later stage. Liver tissue biopsy after treatment shows that administration with high-dose FNC or Lamivudine has the similar effect on improving the inflammatory situation.The results show that FNC can inhibit DHBV replication in vivo significantly.The study provides scientific evidence for the further research which FNC may be a new drug to anti-HBV and could cure hepatitis B effectively.