Molecular Characteristics of Occult Hepatitis B Virus from. Blood Donors in the southeast China

Transcription

1 JCM Accepts, published online ahead of print on November 00 J. Clin. Microbiol. doi:./jcm.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Molecular Characteristics of Occult Hepatitis B Virus from Blood Donors in the southeast China Quan Yuan, a, Shan-Hai Ou, b, Chang-Rong Chen, b,* Sheng-Xiang Ge, a Bin Pei, b Qing-Rui Chen, a Qiang Yan, a Yong-Cai Lin, b Hong-Ying Ni, b Cheng-Hao Huang, a Anthony E. T. Yeo, a James. W.K. Shih, a Jun Zhang, a,* and Ning-Shao Xia a Q.Y. and S.O contributed equally to this work. All authors declare no any conflict of interest. a National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen, Fujian Province, China b Xiamen Blood Service, Xiamen, Fujian Province, China Running Title: Molecular Characteristics of Occult HBV in China * Corresponding authors: Chang-Rong Chen, Xiamen Blood Service, Hubin South Road No.1, Xiamen, Fujian Province 0, People s Republic of China. Phone: Fax: for Chen: ccr1@.com. Jun Zhang, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Siming South Road No., Xiamen, Fujian Province 0, People s Republic of China. Phone: Fax: for J.Zhang: zhangj@xmu.edu.cn Abstract: words. Text: 0 words. 1

2 Abstract The characteristics of 0 carriers with occult hepatitis B virus infection (OBI) were compared with 0 HBV carriers diagnosed at blood donation, 0 asymptomatic carriers and 0 chronic hepatitis patients. The prevalence of genotype C was significantly higher in OBIs than in any other HBsAg+ group (p<0.001). Specific amino acid substitutions in aa-aa1 and aa-aa regions located in the Major Hydrophilic Region of S gene were associated with OBI (p<0.01 for OBI vs. HBsAg+ donors, OBI vs. HBsAg+ asymptomatic carriers and OBI vs. HBsAg+ chronic hepatitis groups). GR was the major variant in the local occult HBV infections

3 Introduction The introduction of hepatitis B surface antigen (HBsAg) screening has reduced the risk of overt HBV transfusion-transmitted infections (TTIs) although this is continuing to occur. In particular, occult hepatitis B virus infection (OBI) poises a threat to the blood supply. OBI is defined as detectable viral DNA in liver or blood by nested PCR or real time PCR, but undetectable HBsAg in serum with current commercial HBsAg assays. Occult HBV status can be associated with mutant viruses undetectable by current HBsAg assays (, 1, 1, ) but may also be due to suppression of viral replication and gene expression and virus secretion (,,, ). The presence of OBI can occur after recovery from infection with anti-hbs being present (1,, ) or anti-hbc may be the only marker () or even with the state where no antibody makers may be present. The presence of HBV is detectable only if a highly sensitive method is used (). The viral load is mostly below IU/ml (), often below 00 IU/ml (). Indeed individuals with anti-hbs or anti-hbc have been shown to be infectious in immunosuppressed organ or bone marrow transplant recipients. To prevent such infections, anti-hbc screening is not 0% effective. Additionally, pooled donation screening, a common practice in blood banks also decreases the sensitivity of the assays used. Finally, if HBV NAT is used, it needs extremely sensitive to eliminate HBV DNA-containing units (1). In HBV endemic countries such as China, research into OBI is limited by the lack of a suitable method for screening large numbers of samples. HBV nucleic acid testing (NAT) is procedurally cumbersome and incurs high costs. Thus given these

4 constraints, using anti-hbc alone' (serum antibodies against HBV core antigen in isolation) as a marker for OBI is investigated in this study given that it may be a possible marker of infection () with one study citing an OBI infection rate of between ~% in patients who tested positive to anti-hbc as the sole marker of HBV infection(). The clinical epidemiology of blood donor OBIs is not known in China and this study attempts to determine the clinical and molecular characteristics of these infections in the context of other cohorts of HBV carriers. Materials and Methods Serological tests for study subjects. 11 blood samples were collected from blood donors at the Xiamen Blood Service, Fujian province, China from July 1, 00 to August, 00. All samples were initially tested negative by rapid HBsAg test. This was a colloidal gold immunoassay with a low detection limit of IU (Wantai Ltd, Beijing, China). To minimize false negative results, three different commercial assays (Murex V ELISA, Abbott Murex, Dartford, UK; Wantai ELISA, Wantai, Beijing, China; Xinchuang ELISA, InTec, Xiamen, China) were used for further HBsAg screening. Samples with positive result in any one assay were be considered as HBsAg-positive. For specimens that were HBsAg negative, additional commercial HBsAg assays (Hepanostika HBsAg Ultra, BioMerieux, Marcy l'etoile,france; Monolisa Ag HBs Ultra, Bio-Rad, Marnes La Coquette,France) were used to confirm the HBsAg status of blood samples. The sensitivity of the five HBsAg assays ranged between 0.0 to 0. IU/mL. HBeAg, and anti-hbs and anti-hbc testing were

5 performed by ELISA (Wantai company, Beijing, China). Anti-HIV, anti-tp (syphilis) and anti-hcv were detected with Murex s ELISA products. All assays followed manufacturer's instructions and were performed on Hamilton ELISA STARlet automated system (Hamilton, Bonaduz, Switzerland). HBV DNA analyses. All anti-hbc alone specimens obtained were tested for HBV DNA. Viral DNA was extracted from 00 µl of plasma using QIAamp DNA Blood Kit (QIAGEN, Hilden, Germany). Six different primers pairs (Table 1) were utilized using nested-pcrs (following the procedures listed in the footnote of Table 1) with all positive cases being retested. Samples that had a confirmatory positive PCR result and a negative HBsAg result were determined to be OBIs. Viral load was determined by real-time PCR (Kehua company; Shanghai, China). Positive PCR products were sequenced on ABI Prism 0X automatic genetic analyzer (Applied Biosystem, California, USA) and phylogenetic analyses performed using neighbor-joining method (MEGA V.1). Control cohort. The control cohort consisted of a group of HBsAg-positive individuals matched only by age and gender with the study group. Among this control group, 0 were prospective blood donors diagnosed as having HBV, 0 were asymptomatic carriers and 0 had chronic hepatitis, all specimens collected from the Xiamen Center for Disease Control and Prevention, Xiamen, China. HBV serologic markers, viral load and HBV genotype were analyzed. The genetic diversity of MHR in S gene was assayed and compared with the OBI donors. Statistical evaluation. Statistical analyses were performed using

6 Mantel-Haenszel χ test and Fisher s exact test for categorical variables, and Mann-Whitney test analysis of variance were used for continuous variables (Open Source Epidemiologic Statistics for Public Health, OpenEpi Version., Differences were considered to be statistically significant for p values 0.0. Demographic data included age, gender and place of birth. The latter is included because the HBV prevalence varies geographically (0). Results Of the 11 samples, 0 tested negative to HBsAg (1 rapid test, and ELISA tests) and 1 samples tested positive to HBsAg. In the 1 samples, only samples were positive in all assays, 0 samples were positive in any two assays and sample was only positive in one assay. Of the 0 HBsAg-negative samples, were anti-hbc alone and of the samples were HBV DNA positive but negative by all commercial HBsAg assays(1 rapid assay, ELISA assays). Thus, these are OBIs, negative in all HBsAg assays (1 colloidal gold immunoassay, ELISAs). In the all OBIs, primer set A had the highest sensitivity and using this primer revealed 0 of positive cases (%), while 1 cases(%),1 cases(%), cases(%), cases(1%) and cases(1%) were positive in primer set Vq, C, S, P and Hc. All OBI samples were clinically asymptomatic, HCV/HIV/TP-free and had levels of ALT below 0 IU/L. Four of the cases were excluded for sequencing because cases tested positive in primer set C, and cases tested positive in primer set P, and these two primers were not targeted at the S gene which primer set A is

7 targeted at. The results of sequences in the MHR region of S gene are shown in Table1. Table compared the birthplace, ALT level, virus load, HBeAg-status, the frequency of genotype C and mutations located the MHR of S gene among 0 OBI identified, and 0 individuals, of the 1 HBsAg test positive individuals above, diagnosed as HBV carriers during blood donation testing ( HBsAg+ donors ), 0 asymptomatic carriers not from the blood donor pool and 0 chronic hospitalized hepatitis patients. The viral load was significantly lower among OBI donors than among asymptomatic carriers or chronic hepatitis patients (p<0.001, p<0.001, respectively), but compared with HBsAg+ donors was insignificant (p=0.0). Phylogenetic analysis indicated that 1 sequences (.% (1 of 0), %CI :.~0.1%) in OBIs, sequences (.0% ( of 0), %CI:.1~.%) in HBsAg+ donors, sequences (1.% ( of 0), %CI:.~0.%) in asymptomatic carriers and 1 sequences (.0% (1 of 0), %CI:.~.%) in chronic hepatitis patients clustered with HBV genotype C. The remaining sequences clustered with HBV genotype B. The prevalence of genotype C s was significantly higher than genotype B (p<0.001) in OBIs. The amino acid map of S protein MHR (aa1-aa1) is shown in Figure 1 stratified by HBsAg status. In the a epitope (aa-aa), the average aa diversity was significantly higher in the OBIs group than any other control group ( p=0.0, OBI vs. HBsAg+ donors; p=0.0, OBI vs. asymptomatic carrier group; p=0.0, OBI vs. chronic hepatitis group, respectively) excluding aa known to be responsible

8 for the determination of serotype. In the region of aa1-aa, the OBI group showed a significantly higher variability than any other HBsAg+ group (p=0.00 OBI vs. donors, p=0.00 OBI vs. asymptomatic carriers, p<0.001 OBI vs. chronic hepatitis patients, respectively). A similar mutation prevalence was observed among each group (Table 1) in the region aa-aa1. There were no insertions or deletions were detected in this region. Specific amino acid substitutions for OBI s were concentrated in the aa-aa1 (p=0.00, OBI vs. HBsAg+ donors; p<0.001, OBI vs. HBsAg+ asymptomatic carriers; p<0.001 vs. HBsAg+ chronic hepatitis, respectively) and aa-aa (p=0.001, OBI vs. HBsAg+ donors; p<0.001, OBI vs. HBsAg+ asymptomatic carriers; p<0.001, OBI vs. HBsAg+ chronic hepatitis, respectively) indicated by red broken lines in Fig 1. Seven (.%, %CI:.~.%) GR mutants, single point or multi-point, were found in OBI group, whereas none were found in the other groups (p<0.001, OBI vs. asymptomatic carriers or chronic hepatitis; p=0.00, OBI vs. donors). Discussion The prevalence of HBV occult infections among non-a-e chronic hepatitis cases is a function of several parameters: (1) the method of detection, ELISA, PCR, real time PCR, () if PCR based, the primer selection will affect the sensitivity and specificity of the test. In our study, primer sets targeting different regions e.g. S, P, C/PreC yielded a sensitivity of between 1%~% in the OBI cohort; () the population being studied, e.g. the prevalence of blood donors with anti-hbc is likely

9 to be very different from the prevalence of HBV DNA found among patients with chronic hepatitis; () patients from HBV endemic areas are more likely to have HBV occult infections if only because of the high numbers of infected individuals present; () the materials being tested e.g. liver or serum. Occult HBV infections are more likely to be found in liver specimens than in serum specimens. OBI prevalence varies greatly. A study from Hong Kong reported a.% prevalence rate while in Italy, the reported rate was %. In Canadian Inuits, it was.1% in subjects devoid of any HBV markers (). In northeast China, the prevalence of OBIs in IgG anti-hbc-positive subjects were 0% (/),.% (/) and.% (/) in cryptogenic chronic liver disease patients, HBsAg-negative HCC patients and HBsAg-negative healthy people, respectively. In these cases, viral load was low (< viral copies/ml) (). In Taiwan, a study using HBV NAT testing yielded cases out of seronegative donations (0.%) (1). The rate reported in this paper was out of 0 HBsAg-negative donations or 0.1%. Detection is thus dependent on a number of factors. In our study, OBI donors averaged 0 years, had normal ALT-level, undetectable HBeAg and very low viral load. There were more males in the group (n=, M:F=1.:1 ) that tested positive to anti-hbc only than either total HBsAg-negative donors (n=0, M:F=1.:1, p<0.001) or HBsAg-positive donors (n=1, M:F=1.1:1, p=0.00). The HBV genotype associated with clinical symptoms and disease progression was genotype C, and this is a common genotype in East Asian countries that has been linked with a higher risk for advanced hepatic disease, such as

10 hepatocellular carcinoma (, 1). The aa substitutions in S protein MHR were found be different in OBIs from HBsAg+ strains. The aa substitutions in aa-aa1 and aa-aa present in OBIs (p<0.001) but not in HBsAg+ strains. It is interesting that these sequences are in the region (aa-aa) which may interfere with hepatitis D virus (HDV) infectivity (). HDV particles are coated with the same envelope proteins (large, middle and small surface antigen) of the HBV (reference). HDV is thus considered an occasional satellite of HBV, because its capacity to propagate depends on the envelope proteins of the latter (reference). If the potential role of aa-aa in infectivity of HDV is confirmed in HBV, the high frequency of mutations in the region might participate in the mechanism of OBI occurrence. Amino acid residues at positions 0 to were considered as an essential for the antigenicity of HBsAg in previous studies (). The mutations within aa-aa,such as GR,DA, were reported as the common escape mutants interfering with HBsAg detection and/or evading vaccine-induced neutralizing antibodies (, 0). Thus, mutations in these two regions may have played a crucial role in OBI occurrence. In conclusion, HBV genotype C and the specific mutations in S gene MHR region were associated with the occurrence of occult HBV infection. The clustering of substitutions in the regions that change the antigenicity of HBsAg and/or the virus infectivity, may play a key role in the establishment and/or maintenance of occult HBV infection.

11 Acknowledgement This work was supported by grants from the excellent Youth Foundation of Fujian Scientific Committee (00J000),National Advanced Technology Research and Development Program (No. 00AA0Z), the Program for New Century Excellent Talents in University (NCET-0-0) and the Key Scientific and Technological Project of Fujian Province (No.). References 1. Allain, J. P. 00. Occult hepatitis B virus infection. Transfus Clin Biol :1-.. Blum, H. E., E. Galun, T. J. Liang, F. von Weizsacker, and J. R. Wands.. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication. J Virol :-.. Busch, M. P. 00. Should HBV DNA NAT replace HBsAg and/or anti-hbc screening of blood donors? Transfus Clin Biol :-.. Candotti, D., P. Grabarczyk, P. Ghiazza, R. Roig, N. Casamitjana, P. Iudicone, M. Schmidt, A. Bird, R. Crookes, E. Brojer, M. Miceli, A. Amiri, C. Li, and J. P. Allain. 00. Characterization of occult hepatitis B virus from blood donors carrying genotype A or genotype D strains. J Hepatol :-.. Carman, W. F., A. R. Zanetti, P. Karayiannis, J. Waters, G. Manzillo, E. Tanzi, A. J. 0 Zuckerman, and H. C. Thomas.. Vaccine-induced escape mutant of hepatitis B 1 virus. Lancet :-.. Chan, H. L., A. Y. Hui, M. L. Wong, A. M. Tse, L. C. Hung, V. W. Wong, and J. J.

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16 Table 1. Different primer pairs of in-house nested-pcr assay a for HBV DNA Primer name Sequences('-') Position(nt) Product length(bp) Target region 1st set primer A sence AF1 '-GTCTGCGGCGTTTTATC-' 1- antisense AR1 '-ACAGTGGGGGAAAGC-' - nd set primer 0 S Hc Vq C P sence AF '-TGCCCGTTTGTCCTCTA-' 0-1 antisense AR '-AGAAACGGRCTGAGGC-' -0 1st set primer sence S1 '-CCTGCTGGTGGCTCCAGTTC-' - antisense S '-ATACCCAAAGACAAAAGAAAA-' -0 nd set primer sence S '-GCGGGGTTTTTCTTGTTGAC-' 0- antisense S '-GGGACTCAAGATGTTGTACAG-' - 1st set primer sense AbF '-CTGCTATGCCTCATCTTCT-' - antisense AdR '-AGGAGTTCCGCAGTATG-' 0-1 nd set primer sence AaF '-CAAGGTATGTTGCCCGTT-' -0 antisense AcR '-ACAAATKGCRCTAGTAAACT-' -1 1st set primer sence AfF '- CAGAGTCTAGACTCGTGGT-' 0- antisense AbR '-GAGAAACGGRCTGAGG-' -0 nd set primer sence AeF '- CCCMAAYCTCCARTCACT-' - antisense AaR '-GAYGATGGGATGGGAA-' 1-1st set primer sence '-GCTTTGGGGCATGGACATTGACCCGTATAA-' - antisense 0 '-CTGACTACTAATTCCCTGGATGCTGGGTCT-' 0-01 nd set primer sence 1 '-GACGAATTCCATTGACCCGTATAAAGAATT-' 1- antisense 01 '-ATGGGATCCCTGGATGCTGGGTCTTCCAAA-' st set primer sence 0F '-CCTATTGATTGGAAAGTATGTCA-' 0- antisense 0R '-AGAATGTTTGCTCCAGACC-' 0-1 nd set primer Small S gene aa1~aa1 Small S gene aa~aa0 Small S gene aa~aa Small S gene aa~aa10 PreC/C gene sence 0F '-CCTATTGATTGGAAAGTATGTCA-' 0- antisense R '-AGTATGGATCGGCAGAGGAG-' - 0 P gene aa~aa1 a: The first round of PCR was performed using an outer primer set for cycles ( C for 0s, C for 0s, and C for 0s). The second round was performed using an inner primer set for cycles ( C for 0s, C for 0s, and C for 0s) followed by the extension reaction. Nested PCR products were subjected to electrophoresis on a % agarose gel, stained by Sybr Green, and visualized using a UV transilluminator.

17 Table. Comparison of characteristics between the occult HBV cases and other HBV groups HBsAg Status Negative Positive P value (OBIs) (control group) Group Identified HBsAg+ ASC Chronic P A,P B,P C b by testing donors a carriers hepatitis (N=0) (N=0) (N=0) (N=0) Age(Mean±SD) 0.±. 0.±. 0.0±. 0.±. Matched Sex(M:F) 1 : 1 : : 1 : 1 Matched Birthplace(Local) (.) (.) (.) (.) 0.,0.1,0. HBV DNA (log copies/ml).±0..±0..0±.0.±1. 0.0,<0.001 *,<0.001 * ALT level(u/l) all below 0 all below 0 all below 0 ± NC c, NC c,<0.001 * HBeAg positive 0(0) 0(0) (1.) (0.0) NC c,0.0 *,<0.001 * Genotype C 1(.) (.0) (1.) 1(.0) <0.001 *, <0.001 *, <0.001 * No. of cases with mutations located in the MHR of HBsAg a epitope aa- (.) (.) (.) (.) 0.0 *,0.0 *,0.0 * AA-AA (.) (.) (.) (.) 0.,0.,0. AA-AA (0.0) 0(0) 0(0) 0(0) *,<0.001 *,<0.001 * GR in a epitope (.) 0(0) 0(0) 0(0) 0.00 *,<0.001 *,<0.001 * AA 1- (.) 0(0) (.) 1(1.) 0.00 *,0.00 *,<0.001 * AA-AA1 (.) 0(0) 0(0) 0(0) 0.00 *, <0.001 *,<0.001 * AA -1 (.) (.) (.) (.) 0.,0.,0. Data are no.(%) or Mean±SD, an asterisk represents a statistical difference of P <0.0. a Diagnosed as HBV carriers during blood donation testing. b P A Value for OBI donors vs.hbv carriers diagnosed during blood donation testing; P B Value for OBI donors vs. asymptomatic HBV; P C Value for OBI donors vs. chronic hepatitis. c NC: Non-calculated.

18 Figure Legends Fig 1. Amino acid variability of S protein MHR (aa1-aa1) in occult HBV and control cases. Consensus amino acid sequence of genotype B and C were deduced from AB0 and AF, respectively. Strains whose amino acid sequence was identical to the wild type are not represented in the figure. These non-represented strains include the following: OBI: -,-, Donor: 1, -, -, 1-1, 1,, -, 0 Asymptomatic chronic carriers: -, -, -, 1, 1-, -,, 1-,, 1, -, -, 1,, -0 Chronic hepatitis: 1, -,,, 1, 1-0,, -, 1-, -0, -,,, 0 (genotype B, adw) OBI: 0,,, - Donor: 1,, Asymptomatic chronic carrier:,,, 1,,,,, Chronic hepatitis:,,, -, 1,,, (genotype C,adrq+)

19 Occult HBV infections(donors) Overt HBV infecitons(control cohort) "a" epitope aa-aa Genotype B I P G S S T T S T G P C K T C T T P A Q G T S M F P S C C C T K P T D G N C T C I P I P S S W A F A K Y L W E W serotype OBI R adw OBI I adw OBI T adw OBI Y V adw Genotype C L P G T S T T S T G P C K T C T I P A Q G T S M F P S C C C T K P S D G N C T C I P I P S S W A F A R F L W E W serotype OBI-,1, R adrq+ OBI, R adrq+ OBI I P R - R adrq+ OBI I R R adrq+ OBI1, T - R A ayr OBI G V R adrq+ OBI R adrq+ OBI K adw Genotype B I P G S S T T S T G P C K T C T T P A Q G T S M F P S C C C T K P T D G N C T C I P I P S S W A F A K Y L W E W serotype Donor V - adw Donor,CH N - S F adw Donor,,, F adw Donor N F S L adw Donor N S S adw Donor S R adw Donor,ASC T adw ASC T L adw ASC I adw ASC1,,,CH A adw ASC L adw ASC T T adw ASC I A - adw ASC T T L adw CH L G - adw CH R adw ASC,CH R ayw CH R I ayw CH R G - ayw CH1, R adw CH H adw CH I adw Genotype C L P G T S T T S T G P C K T C T I P A Q G T S M F P S C C C T K P S D G N C T C I P I P S S W A F A R F L W E W serotype ASC A adrq+ CH K adw CH A adrq+ Abnormal amino acid mutation Genotype/subtype-related amino acid variability Significant different region between occult and control cases