| BackgroundHepatitis B virus(HB V)infection is a global health problem.HBV DNA can still be detected in liver tissues or serum of HBsAg negative patients.This phenomenon is called occult hepatitis B virus infection(OBI).OBI is a special form of hepatitis B infection that can lead to acute exacerbation of hepatitis B,cirrhosis and hepatocellular carcinoma(HCC).HBsAg is negative and HBV DNA is often at a very low level in OBI serum,which makes it difficult to diagnosis in blood screening and clinical.The pathogenic factors of OBI are complex,and its mechanism has not been fully studied.HBV gene mutation,especially the major hydrophilic region(MHR)and "a" determinant mutation in S region,is one of the mechanisms leading to OBI.The purpose of this study is to identify and verify the mutation sites related to OBI in S region,and to further elucidate the pathogenesis of OBI.Object123 OBI donors collected in Blood Center from April 2015 to May 2017MethodsNucleic acid extraction,viral load determination and PCR gene amplification were performed in OBI donors,and nucleotide sequence of S region was determined.The S-region sequence characteristics of OBI and HBsAg+/HBV DNA+blood donors(control group)were analyzed.Amino acid sequence and OBI-related mutations of MHR,"a" determinant and S region outside of MHR(NMHR)were analyzed between and within groups.In order to further verify the OBI B genotype-related mutation sites,a conservative full-gene expression vector of HBV B genotype was constructed.A mutant plasmid containing OBI-related mutation sites was constructed by site-directed mutagenesis and transfected into HepG2 cells to analyze the expression of extracellular and intracellular HBsAg level.The distribution of HBsAg in some mutant plasmids transfected cells was observed by immunofluorescence staining.Using GraphPad Prism 7.0 statistical analysis software,Fisher exact test analysis of classification data between two groups,Mann-Whitney U test analysis of continuous variable data,Fisher's LSD test was used to compare the relative expression of HB sAg in the mutant plasmids.All statistical tests were carried out by bilateral test,P<0.05 was statistically significant.Results1.The number of amino acid mutations in MHR,"a" determinant,NMHR and S protein of OBI B genotype and C genotype were significantly higher than those of control group(P<0.001),indicating that there was significant variation in OBI S region.The number of mutation rates in MHR and "a" determinants of OBI B genotype and C genotype was significantly higher than that of NMHR(P<0.05),However,the HBV B genotype control group had the opposite result:The mutation rates of MHR and "a" determinant were lower than those of NMHR in HBV B genotype control group(P<0.05).The mutation rates of MHR were lower than those of NMHR in HBV C genotype control group(P<0.05).The results showed that the mutations of MHR amino acids in OBI group were significantly higher than those in NMHR group,while in wild type HBV,MHR was arelatively conservative region.There was no significant difference in the number of amino acid mutations of S protein between OBI B and C genotypes(P>0.05).2.Ten mutations in OBI B genome(E2G,Q101R,K122R,M133T,D144E,G145R,V168A,S174N,L175S and I226S)significantly frequency than those in the control group(P<0.05).Twenty-eight mutations(E2G,T4I,F8L,V14A,L98R,Q101K,Q101R,M103I,S114T,T116A,S117G,S117N,T118K,T118R,K122R,P127T,A128V,Q129P,M133I,S136Y,D144E,G145A,R160K,E164G,V168A,S174N,L175S and M198I)in OBI C genome were significantly frequency than those in the control group(P<0.05).Because E2G,Q101R,K122R,D144E,V168A,S174N and L175S are OBI related mutations both found in B and C genotypes,there were 31 mutations in the OBI group were found to be significantly frequency than those in the control group(P<0.05).The frequency of OBI related mutations was 8.9%-30.0%in the OBI sequence,while in the control group,the frequency of these mutations was only 0%-3.8%.Of the 31 OBI related mutations,15 were reported in previous studies(Q101R,Q101K,M103I,S114T,S117G,T118K,K122R,P127T,Q129P,M133T,D144E,G145R,G145A,S174N and L175S),and 16 were not reported before(E2G,T4I,F8L,V14A,L98R,T116A,S117N,T118R,A128V,M133I,S136Y,R160K,E164G,V168A,M198I and I226S).3.Cell transfection experiments showed that HBsAg expression was detected in the supernatant(extracellular)and lysate(intracellular)transfected with 10 mutant plasmids of HBV B gene and wild-type plasmids(>1COI).Compared with pHBV 1.3B,the relative levels of extracellular HBsAg in E2G,D144E,G145R,V168A and S174 decreased by 96.2%,39.4%,84.2%,77.7%and 37.4%(P<0.05),and the relative levels of intracellular HBsAg in E2G and M133T increased by 139.3%and 51.8%(P<0.05),while the relative levels of intracellular HBsAg in G145R decreased by 48.11%(P<0.05).It is noteworthy that extracellular HBsAg(3.8%VS pHBV 1.3B)and intracellular HBsAg(239.3%VS pHBV 1.3B)with E2G mutation are significantly different from those with pHBV 1.3B(P<0.0001),showing obvious characteristics of blocked HBsAg secretion.Other mutations(Q101R,K122R,M133T,L175S and I226S)had no significant effect on intracellular and extracellular HBsAg detection(P>0.05).4.Immunofluorescence results showed that HBsAg expression was detected in cells transfected with 10 different mutant plasmids and pHBV 1.3B of HBV B gene.In pHBV 1.3B plasmid transfected cells,HBsAg fluorescence was widely distributed in the whole cytoplasm and dispersed uniformly in fine particles.In contrast,HBsAg showed two different distribution types in cells transfected E2G mutant plasmid.Most of the cells showed high-density distribution near the nucleus or even the whole cytoplasm,while a few cells showed coarse granular distribution in the cytoplasm.The fluorescence optical density of E2G expressing HBsAg(median,0.091 IOD/pixel)was significantly higher than that of pHBV 1.3B(median,0.043 IOD/pixel)(P<0.0001).Conclusions and significances1.There was significant variation in OBI S region.The mutation of MHR in S region may play a more important role in the formation of OBI.2.Thirty-one representative OBI related mutations were found in the OBI group,among which E2G,T4I,F8L,V14A,L98R,T116A,SI 17N,T118R,A128V,M133I,S136Y,R160K,E164G,V168A,M1981,and I226S were novel OBIrelated mutations.3.In the OBI B genotype,E2G,D144E,G145R,V168A and S174N can affect the detection of HBsAg at the cellular level.E2G and V168A are new mutations,and extracellular experiments have confirmed that they have significant effects on the expression of HBsAg.The mechanism of OBI induced by E2G mutation is the accumulation of HBsAg in cells and the decrease of HBsAg secretion due to the disorder of HBsAg secretion. |
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