| Objective:Hepatitis B virus (HBV) is one of globally prevalent pathogens which cause human hepatitis. Although the HBV prevalence in our country decreased from9.75%in1992to7.18%in2006, there are still93millions of chronic HBV carriers. At the mean time, approximately20millions of patients have chronic viral hepatitis. About300,000patients died from liver cirrhosis and liver cancer each year. The newly infected cases are an half to one million. Previous studies have found that the risk of transfusion transmission of HBV are significantly higher than other viruses, which are mainly from the window period infection, mutant virus strains and occult HBV. The HBsAg negative but HBV DNA positive (HBsAg-/DNA+) infection is known as occult hepatitis B virus infection (OBI). In2008, OBI was defined as the individuals were detected HBsAg negative but HBV DNA positive in their liver by the current available methods, while the HBV DNA cannot be detected in the serum or plasma. However, it is difficult to detect the DNA in the liver at clinical practice. Therefore, OBI is now defined as the individuals have HBV DNA, but not HBsAg in blood, while anti-HBc or anti-HBs are either positive or negative.According to the diversity of HBV genome nucleotide sequence differences≥8%or S gene sequence≥4%, HBV has been divided into8genotypes A-H, of them each genotype has been sub-divided into several subtypes. HBV genotypes are obviously geographic distribution. Northern and Western Europe and the USA primarily have genotype A, while genotypes B and C are mainly distributed in Asia, D is most widely distributed in the world. The genotype C is dominant strains in north China, B is more common in southern region. The mechanisms for occurrence and evolution of OBI strains are not fully clear. Possible mechanisms are:(1) HBsAg and anti-HBs form to immune complexes;(2) variant affects its antigenic determinant;(3) co-infection with other hepatitis viruses affects HBV replication and reduces HBsAg. Detection of HBsAg is currently used as the indicator of HBV infection, while nucleic acid test (NAT) has not been implemented in blood screening. The residual risk of HBV, OBI and window period infection still exist in blood transfusion.The characteristics of OBI of genotypes B and C are not completely understood. In this study, we molecularly characterized OBI B and C strains including (a) the prevalence of OBI in Shenzhen;(b) the molecular biological characteristics. The data obtained will provide an important information for improving assays for blood safety screening, and provide theoretical guidance for clinical treatment of OBI.Materials:310,167blood samples were collected from donors between April2010and December2012in Shenzhen Blood Center, in which114,761(37%) are the first-time donors, and195,406(63%) are repeat donors. All plasma samples with negative HBsAg and normal ALT detected by rapid tests with the colloidal gold dipstrips were screened by enzyme immunoassay (EIA) and nucleic acid test (NAT) for HBV HBsAg-/DNA+samples.Methods:(1) Blood donors specimens were detected by two different assays of imported and domestic kits for screening of HBsAg, and then followed for detection of HBV DNA by the NAT (Novartis TIGRIS, the United States). HBsAg-/DNA+samples were confirmed by highly sensitive nested PCR and QPCR and chemiluminescent microplate immunoassay (CMIA).(2) HBsAg-/DNA+plasma samples were quantified for HBV DNA loads by QPCR.(3) Basic core promoter/pre-core (BCP/PC), full-length genome, pre-S/S, S and core regions were cloned and sequenced.(4) Full length sequences of wild type HBV strains of genotypes B and C were retrieved from the GenBank database as reference sequences. S region of OBI strains was phylogenetically analysed for genotypes by MEGA.(5) The sequences of whole genome, pre-S/S, P, X and Pre-C/C regions were analyzed. Deduced amino acid sequences of each region were compared to corresponding sequences of HBV wild-type strains obtained from the GenBank database.(6) The mutations were characterized including regulatory elements including promotes (SP1,SP2, CP, XP), regulatory sequences (core upstream regulatory sequence, CURS, DR1, DR2, negative regulatory element), enhancers (ENH1and EENH2).(7) SPSS software (version13.0) was used for statistical analyses. Categorical variables were compared by using Pearson Chi-Square test to compare the differences between OBI and wild-types strains on virus protein and regulatory sequences. Mann-Whitney U test was used for continuous variables comparison of HBV DNA loads between of OBI and HBsAg+samples. Statistical significance was defined as P<0.05(two-tailed).Results:1.121HBsAg-/DNA+plasma samples were classified, in which6samples (SZ81, SZ82, D1, D3, D7, D17) were false positive,4samples (SZ7, SZ10, SZ31, SZ33) became HBsAg positive,12samples (SZ16. SZ21, SZ22, SZ54, SZ55, SZ80, D5, D6, D10, D15, D21) were negative for all five serological markers, and final99HBsAg-/DNA+samples were defined as OBI. The yield of OBI infection in Shenzhen was99/310167. The53genotype B and13C were identified. This study obtained20whole genomes (17type B,3type C),31Pre-S/S (25genotype B,6genotype C),18S (15genotype B,3genotype C),55Pre-C/C (45genotype B,10genotype C) sequences from OBI samples. According to S sequence,57genotype B and12genotype C were analyzed from99OBI samples. Of99donors with OBI, there were72males and27females who were all with normal ALT levels (<40). By clarifying with serological markers,99OBI samples were classified into31(31%) HBcAb+/HBsAb+, with viral load range1-798.7IU/ml (median61.81IU/ml),12(12%) HBsAb+/HBcAb-with viral load range1-1205IU/ml (median63.87IU/ml),53(54%) HBcAb+/HBsAb-,with viral load range1-1062IU/ml (median53.86IU/ml),3(3%) HBcAb-/HBsAb-with viral load range13.33-550.6IU/ml (median101.2IU/ml), respectively. The ages of OBI donors ranged between20and56years old (median35years), viral loads ranged between un-quantifiable (<1IU/ml) and2122IU/ml (median27.9IU/ml).2. The regulatory element sequences of OBI were analyzed.(1) DR1, DR2and NRE regions within OBI genotype B and C were highly conserved;(2) SP1, SP2, ENH1, ENH2regions within OBI genotype B and type C were more frequently mutated than the regions within wild-type (P<0.05);(3) CURS within OBI genotype B had higher nucleotide mutation rates than wild-type strains (P=0.037), while BCP within OBI genotype C had higher nucleotide mutation rates than wild-types of HBV (P=0.002).40%of BCP within OBI genotype B had mutation at nt1752,38%CURS mutated at nt1726.31%OBI genotype C mutated at nt1799,46%CURS within OBI genotype C mutated at nt1727, but no deletion and insertion occurred and no mutations at nt1762and nt1764nt were found.3. The protein sequences of OBI were analyzed.(1) The mutation of Pre-S/S amino acid (aa) sequences within OBI genotypes B and C was significantly higher than wild types of HBV (P<0.0001).The mutation of Pre-S1aa sequence within OBI genetype C was higher than wild type strains (P<0.0001), while Pre-S2and S mutations within OBI genotype B were higher (P<0.05). Mutation in MHR sequence occurred frequently. Mutation frequencies of F134I/L and G130R/A concentrated in aa130-134occured13(18%) and9(12%) times within OBI genotype B sequences, respectively. Samples SZ5, SZ98and D11had an insertion of SPPTGTGS, IPGSPPTG and PR at aa121,118and124in the MHR, respectively. Samples SZ97had a stop codon at aa185;(2) The diversity in P aa sequences of OBI genotypes B and C was higher compared with wild type of HBV (P<0.001). The important motifs YMDD and FLLA are highly conservered in OBI samples.47%of OBI genotype B samples had the mutations of K73N, V458D/A, and A568T, while three OBI genotype C samples had E73D mutation. Sample SZ5had a9amino acid insertion of NSRTITTNP at position aa471. While SZ15had an insertion of TQ at aa477.(3)Mutation frequency within Pre-C/C sequences of OBI genotype C was significantly higher than wild type of HBV (P=0.034).29%OBI genotype B samples appeared G1896A mutation,4OBI genotype C samples had these mutations, which led a stop codon at position aa28and stoped the translation of HBeAg. Sample SZ28, SZ39and D14had the deletions at nt1839,1876and1841-1845, which caused reading frame shift and occurrence of stop codon.(4) The mutation within X aa sequence of OBI genotypes B and C sample was more frequently than the wild types of HBV(P<0.0001).Conclusion:In this study,121HBsAg-/DNA+blood samples were detected from310,167blood donors in Shenzhen blood center. Of121samples,99were identified as OBI, in which OBI genotype B was predominant. More than20whole genome and S region sequences were analyzed, which suggested that the mutation occurred more frequently than those of HBV wild types. Higher mutation was observed for Pre-C/C regions, which lead frequent occurrence of termination of amino acid translation affecting core protein production. The results obtained from this study will be helpful for improving blood screening assays for OBI detection and helpful for clinical treatment of patients with OBI. |
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