A Study Of Multidrug Resistance Mutations Of Hepatitis B Virus Genome And Innate Immune Factors In Hepatitis B Patients

Background and Purpose Hepatitis B virus(HBV)is widespread in China and the morbidity in china is 7.18 %.Persistent infection with the HBV causes chronic hepatitis B,cirrhosis of the liver and primary liver cancer,therefore the HBV seriously affects people's health.With the discovery of the nucleotide analogues anti HBV drugs,a part of hepatitis B patients can be effectively treated and cured.With the broad and long-term application of nucleotide analogues,the problem about its multidrug resistance has become a new challenge for the treatment of hepatitis B.In the present study,we will analyze the gene sequence variation loci of multiple drug resistance HBV and try to interpret the molecular mechanism of anti-HBV drug multiresistance to nucleotide analogs.Meanwhile,the m RNA of IFN-?,IFN-?,Mov10 and TRIM22,which are innate immune factors,will be detected in the chronic hepatitis B patients with nucleoside acid analogues multidrug resistance.We will investigate host innate immune status and discuss the host immune molecular mechanism under the nucleotide analogues multiresistant HBV infection.Methods1.Twenty eight patients with nucleotide analogue resistance and twenty healthy individuals in the control group were collected from the department of infectious diseases in Heilongjiang province hospital.2.Whole genome amplification of multidrug resistance HBV DNA in 6 patients was performed by polymerase chain reaction(PCR)and p MD18T-HBV plasmids containing HBV genome were constructed,sequenced,and analyzed to find resistance mutations.3.The m RNA of IFN-?,IFN-?,Mov10 and TRIM22,that are the innate immune factors in peripheral blood,were detected in 28 patients with nucleotide analogue resistance and 20 healthy individuals using RT-PCR and the statistical analysis of these data was performed.Results1.HBV whole genome was amplified in 6 patients with nucleotide analogues multidrug resistance and 19 clones of p MD18T-HBV containing HBV DNA were constructed successfully.2.Comparing with HBV standard strain genotypes,all the isolated multidrug resistance HBV strains were genotype C.3.Based on the sequence analysis of the 19 strains of multidrug resistance HBV gene,we found the common main mutation rt M204V/I in P region which codes the reverse transcriptase(rt),along with the secondary mutations including rt Q333 K,rt H337 N and rt D392S;common main mutations T56 N,K57Q,D62 A and V157 A in S region;common main mutations A41 S,G85A and L92 V in X region;no common main mutation in C region.4.The m RNA levels of Mov10 and TRIM22 that are innate immune factors in peripheral blood PBMC of multidrug resistance chronic hepatitis B patients were higher than those of control groups with statistically significant differences(P <0.01).5.TRIM22 m RNA in chronic hepatitis B patients with multidrug resistance showed positive correlations with m RNA levels of IFN-?,IFN-? and Mov10,respectively;Mov10 m RNA showed positive correlations with m RNA levels of TRIM22 and IFN-?;IFN-? m RNA showed positive correlations with m RNA levels of IFN-?,Mov10 and TRIM22.6.There were no statistically significant differences in m RNA levels of the innate immune factors including IFN-?,IFN-?,Mov10 and TRIM22,between multidrug resistance group and cross-resistance group.Conclusions1.We found several common mutation locis in P,S,X regions of HBV DNA in 19 clones isolated from 6 chronic hepatitis B patients with multidrug resistance.2.In peripheral blood PBMC the m RNA levels of Mov10 and TRIM22,innate immune factors,were higher in the chronic hepatitis B patients with nucleoside analogues resistance than those of the healthy control group.3.The correlation analysis of m RNA levels between IFN-?,IFN-?,Mov10 and TRIM22 demonstrated that the correlations among them were different under the nucleoside analogues resistant HBV infection.