Study On Intrahepatic And Extrahepatic Tissues Infection By Duck Hepatitis B Virus And Its Effect On Glucose Homeostasis

Objective: The incidence of diabetes mellitus in the patients with hepatitis B is significantly higher than that in the normal population.However,the pathogenesis of hepatitis B complicated by diabetes remains unclear.This thesis examined the infection of intrahepatic and extrahepatic tissues by duck hepatitis B virus（DHBV）and its effect on glucose homeostasis using a DHBV infection model.Methods:（1）Whole blood was collected from 97 domestic ducks（Anas platyrhyncha domestica）with the same genetic background and serum was separated,and the total DNA of the duck serum was extracted.Using the total DNA as a template for PCR detection and the DHBV-bearing ducks in the serum were screened.Serum DHBV DNA was detected using the SYBR green quantitative PCR（q PCR）method as described previously.（2）Tissue DHBV DNA was extracted from the tissue samples of liver,pancreas,kidney and skeletal muscle were collected from the DHBV-positive ducks.DHBV covalently closed circular DNA（ccc DNA）was detected by applying amethod of rolling circle amplification（RCA）combined with gap PCR of duck hepatitis B virus replication in intrahepatic and extrahepatic tissues.DHBV DNA content in liver,pancreas,kidney and skeletal muscle tissue was quantified with the q PCR method.（3）fluorescence in situ hybridization（FISH）assay was employed for verifying tissue DHBV infection.（4）Blood glucose concentration was determined by intravenous glucose tolerance test on domestic ducks were fasted for 16 hours.（5）Extracting samples of glucose tolerance test and measuring plasma insulin levels.（6）Statistical analysis was performed with SPSS 18.0software.The levels of blood glucose and plasma insulin at each time point during the GTT were compared between DHBV-infected and DHBV-non-infected ducks by using a one-way analysis of variance（ANOVA）,the effect of DHBV infection on glucose homeostasis.The correlations among the levels of blood glucose and plasma insulin during the GTT and the levels of the DHBV DNA of the plasma and the tissues were examined by the nonparametric Spearman’s rank correlation test.Results:（1）The serum total DNA of 97 domestic ducks was screened by PCR.53 ducks were infected with DHBV congenitally,44 ducks were not infected with DHBV,and the infection rate of DHBV was 54.6%.（2）The liver,kidney,pancreas and skeletal muscle of DHBV positive serum and serum DHBV DNA were detected by applying amethod of rolling circle amplification（RCA）combined with gap PCR to detect covalently closed circular DNA（ccc DNA）,and the detection rates were 100%,75.5%,67.9% and 47.2%,respectively,and the results were verified by in situ hybridization.（3）In a glucose tolerance test（GTT）,significantly higher blood glucose levels were observed at the time points of 15 min and 30 min in the DHBV-infected ducks（P < 0.05）,as compared with that of the DHBV-non-infected ones.Moreover,the higher blood glucose levels maintained up to 2 hours（P < 0.01）during the GTT in the ducks with the infection of skeletal muscle by DHBV ccc DNA.associations were observed among the DHBV DNA levels of both plasma and tissue samples and the levels of blood glucose during the GTT in the DHBV-infected ducks.（4）No significant differences in baseline insulin between DHBV-infected ducks and DHBV-non-infected ducks（P > 0.05）and insulin levels after glucose injection（P > 0.05）.There were no statistically significant correlations between the levels of blood glucose and plasma insulin during the GTT in eitherthe DHBV-infected ducks or the DHBV-non-infectedonesas well as among the levels of the blood glucose and the plasma insulin and the DHBV DNA in plasma and tissues.Conclusions:（1）DHBV-infected ducks have viral infections in liver and extrahepatic tissues such as kidney,pancreas and skeletal muscle,and the skeletal muscle infection by the virus is the first time to be observed.（2）The blood glucose level of the DHBV-infected animals increased significantly during GTT,and the abnormal increase of blood glucose level was related to the infections of both intrahepatic and extrahepatic tissues by the virus.