Development Of Quantitative Evaluation System For DHBV Model And Using In Evaluation Of DHBV Inhibit Effect Of Chinese Herb Complex

About 4000 million people all over the world are chronically infectedwith Hepatitis B virus (HBV), which result in 3 million people with livercancer every year and result in 5 to 1.2 million deaths per year causedby chronic hepatitis, cirrhosis, and hepatocellular carcinoma. About onethird of people in the world had infected by HBV and there are 2—20% of these infected people transmitted to be chronic HBV infection. Sothe researches related to HBV are always the hot point.Both DHBV and HBV are classified to hepadnavirus. There aresimilarities in amplification style, pathogenesis and many other facetsas comparison of the 2 virus. So the duck animal model had been used asthe research model for anti-HBV drug screening and HBV pathogenesis formany years. The dot-blot assay, which is an economic and high-flux assay,had been used in testing DHBV for many years. But the degree of accuracyand sensitivity of the dot-blot assay are not very satisfactory.Additionally, the duration of drug administration is 10 day in common,which is too short for the Chinese herb with the characteristic of milddrug effect.The Duck embryo liver cell model is believed to be the onlyhepadnavirus cell model that cells can be infected by DHBV straightly andthe DHBV can be amplified stably. While there isn't a good evaluationmethod for evaluation of the DHBV load in the supernatant of the duckembryo liver cells infected with DHBV. The dot-blot assay had been usedfor semi-quantitatively test the DHBV in the supernatant of DHBV infectedduck embryo liver cells, but is need 600-800μL supernatant for spottingand can not get a precise result. So this research developed a quantitativeevaluation system for DHBV model and used this method in evaluation ofDHBV inhibit effect of Chinese herb complex. 1 The conserved region in the DHBV genomes were looked up through aligning32 DHBV complete sequences. The primer and probe for quantitative PCR weredesigned within the DHBV conserved region. The DHBV standard plasmid wasconstructed use the DHBV PCR product which included the target sequenceof DHBV quantitative PCR to insert into Pmdl8-T vector. The DHBV standardplasmid was tested by the quantitative PCR method and the results showedthat 8 dilution serials linear correlation with the Ct value. The testingrange is from 2 to 2e7 copy in the 25μL reaction system. The intraassayof the method is≤2.03%; The interassay of the method is≤2.47%.2 Through comparison of NP-40 serum virus DNA extraction method, Sodiumcotanoate serum virus DNA extraction method and a serum DNA extractionkit from DaHui biotechnological corporation, the extraction efficiencywere shown that the NP-40 method was the poorest and the Sodium cotanoatemethod is similar to DNA extraction kit from DaHui biotechnologicalcorporation in extraction efficiency. Because the Sodium cotanoate methodis far more economic than the kit from DaHui biotechnological corporation,the Sodium cotanoate method is the fittest method for quantitative PCR.3 The congenitally DHBV positive ducks from I00 Guangdong brown ducks werescreened with PCR method. These congenitally DHBV positive ducks were usedas animal model for evaluation of the DHBV inhibition effect of waterextraction of the herb complex of strengthening healthy Qi combined withdraining dampness and activating blood (SHQDDAB). The water extractionof the SHQDDAB was administrated for 1 month and the serum samples werecollected at the time points of first day (D0), tenth day (D10), twentiethday (D20), thirtieth day (D30) of drug administration and 5 days postcessation of the drug administration (P5).The DHBV load of these collectedserum were tested with the DHBV quantitative PCR method. The resultsshowed that the serum DHBV load of both the high and low dosage of SHQDDABwere downward at D10, D20, D30. Even in the P5 the DHBV load also downwardin some sense.4 The dynamic changes of the DHBV load in the supernatant of thecongenitally and postnatally DHBV positive duck embryo liver cell culturewere tested with the DHBV quantitative PCR method. The results showed that the highest DHBV load was occurred in the second day after seeding ofcongenitally DHBV positive duck embryo liver cells. Then the DHBV loadwas deeply downward at third day after cell seeding. But the DHBV loadwas gone upward slowly after the third day and ascended to a peak at thesixth day after cell seeding. While the DHBV load of all the time pointsare within 1.21-3.78e8 copies/ml. So it was believed that the DHBV loadin the supernatant of congenitally DHBV positive duck embryo liver cellsare stable in some sense.Similar to the congenitally DHBV positive duck embryo liver cells,the highest DHBV load was occurred in the second day after DHBV infectionand then deeply downward at third day and done upward slowly in theremained days. The peak was occurred at 11 days after DHBV infection. Theseresults are markedly different from the previous results obtained bydot-blot assay. Compared with congenitally DHBV positive duck embryoliver cells, the dynamic range of DHBV load in the supernatant ofpostnatally infected DHBV positive duck embryo liver cells are from 2.98e5to 2.65e8 copy/ml, which is far bigger than the range of DHBV load ofcongenitally DHBV positive duck embryo liver cells.In this research, 20μL, 50μL and 100μL of DHBV positive serum wereadded to the cell culture system respectively for infection. The resultsshowed that the peak of the DHBV load were occurred at the same day (ninthday after DHBV infection) and the DHBV load have no marked differencesafter the fourth day post DHBV infection in all the 3 dosages of DHBVpositive serum infection to duck embryo liver cells.5 The DHBV inhibition effect of ethanol and water extraction of SHQDDABwere evaluated with the congenitally DHBV positive duck embryo liver cellculture system. The drugs were administrated for 5 days and thesupernatant samples were collected every other day. The results showedthat the water extraction of SHQDDABhas no marked inhibit effect to theDHBV in vitro in all the 3 dosages. While the ethanol extraction shownsignificant inhibition effect to the DHBV in the supernatant of thecongenitally DHBV positive duck embryo liver cell culture system. Theseresults revealed that the SHQDDAB may not directly inhibit the DHBV in vivo. It may inhibit DHBV in vivo through promoting the anti-virus immunesystem.The ethanol extraction of SHQDDAB showed significant inhibition ofDHBV in vitro revealed that the ethanol extraction was different from thewater extraction of SHQDDAB and may include some reagents which caninhibit DHBV directly. This may need further research.This research established a complete testing system of quantitativelytesting of DHBV which significantly elevate the sensitivity and precisionof DHBV testing. The DNA extraction method for quantitative PCR alsoestablished and integrated to the DHBV quantitative PCR testing system,which make the DHBV quantitative testing system can be precisely andlarge-scaly application. The duration of drug administration wereextended to 1 month in the duck animal model that is more suitable forDHBV inhibition effect evaluation of Chinese herb. It is the first timethat the dynamic changes of the DHBV load in the supernatant of thecongenitally and postnatally DHBV positive duck embryo liver cell culturewere tested with the DHBV quantitative PCR method. It was revealed thatthe congenitally DHBV positive duck embryo liver cells were more suitablefor in vitro anti-DHBV drug effect evaluation than the postnitallyinfected DHBV positive duck embryo liver cell culture system. By doingof these research works, the quantitative evaluation system of DHBV modelhad been established and the sensitivity and precision of DHBV model waselevated.The DHBV inhibition effect of the SHQDDAB herb complex was evaluatedwith the established quantitative evaluation system of DHBV model. Theresults showed that the DHBV load in the serum of congenitally DHBVpositive ducks was markedly inhibited by the water extraction of theSHQDDAB in one month drug administration. In the in vitro experiments,the water extraction have no marked inhibit effect to the DHBV in thesupernatant. While the ethanol extraction of the SHQDDAB showedsignificantly inhibit the DHBV in the supernatant of congenitally DHBVpositive duck embryo liver cells. These revealed that the SHQDDAB may not directly inhibit the DHBV in vivo. It may inhibit DHBV in vivo throughpromoting the anti-virus immune system.If the testing method for covalently closed circular DNA of DHBV canalso be quantitatively established, the quantitative evaluation systemof DHBV model will be better. The inhibit effect of the herb complexSHQDDAB to DHBV need further research.