Molecular characterization of hepatitis B virus isolates from Zimbabwean blood donors

Hepatitis B virus (HBV) is endemic in Africa, being hyperendemic in sub-Saharan Africa. Genotypes A, D, and E circulate in Africa, showing a distinct geographical distribution. The aim of the present study was to determine the HBV genotype distribution in blood donors from different geographical locations in Zimbabwe. Using a restriction fragment polymorphism assay, sequencing of the basic core promoter/precore region and of the complete S open reading frame showed that 29 HBV isolates from geographically distinct regions belong to subgenotype A1. The complete genome of two of these Zimbabwean HBV isolates was sequenced. Forty-four percent of the Zimbabwean HBV isolates (11/23) were characterized by a G1862C missense mutation, which causes a Val to Leu amino acid substitution at position 17 of the precore region. The majority of Zimbabwean HBV isolates clustered with a number of South African HBV isolates, with which they shared characteristic amino acids in the preS1, preS2, and polymerase spacer regions. The wide distribution of subgenotype in Africa, as well as the high intragroup divergence and the geographical clustering of the African and Asian subgenotype A1 HBV isolates indicate that this subgenotype has a long period of endemicity in these regions.     

Molecular characterization of hepatitis B virus in blood donors in Botswana

Virus Genes - Hepatitis B virus (HBV) poses a significant threat to blood transfusion safety in sub-Saharan Africa (SSA) where allogeneic blood donations are screened serologically, and more...     

Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia (<10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An unusually large number of amino acid mutations was present in the immunodominant a-determinant of HBsAg (respectively 3, 6, 7, 10, and 10 mutations). Comparison of the HBV genomes in two donors to a consensus HBV genotype D sequence showed a most prominent hotspot of genetic variation in HBV nucleotides 480-570, encoding the HBsAg a-determinant. The phylogenetic comparison of separate donor HBV genes to the HBV genes of 11 reference strains (genotypes A-H) showed the donor HBV surface genes to form an outgroup, while the HBV polymerase, core and X genes closely cluster with the HBV genotype D reference strain. Maybe the HBV strains in this study represent a natural end-stage of seemingly cleared HBV infection, in which HBV maintains a low level of possibly non-infectious replication, after sacrificing its immunologically offending surface antigen, thus avoiding final clearance by the immune system.     

Molecular and serological evaluation of surface antigen negative hepatitis B virus infection in blood donors from Venezuela

Surface antigen negative hepatitis B virus (HBV) infection was evaluated in Venezuela, by molecular characterization of blood samples positive for antibodies to core antigen (anti-HBc) and negative for surface antigen (HBsAg) in blood donors (residual infections). HBV DNA was found in 11/258 samples (4.3%), and was significantly associated with high levels of anti-HBc antibodies (>25 UI/ml, P < 0.05), while no correlation was found between the presence of HBV DNA and the levels of anti-HBs. Synonymous and non-synonymous mutations were found in the HBV surface region (but not vaccine escape mutants) and in the precore/core region (precore mutants in 2/7 samples and 33-45 bp deletions near the N-terminal core region in 4/19 samples). While HBV genotype F prevails among HBsAg positive samples from blood donors in Venezuela, residual infection isolates were mainly genotypes A and D. Phylogenetic analysis of viral surface and core region revealed discrepancies in genotype designation in 6/9 samples, suggesting the presence of mixed infection or recombination. In conclusion, HBV residual infection in Venezuela does not seem to be frequently observed in HBV genotype F. This type of infection is frequently associated with variants exhibiting mutations in the surface gene that might be affecting the correct recognition by commercial tests, with precore mutants and with core internal deletions. These variants do not seem to cause severe liver disease, and on the contrary, were found circulating at low viremia.     

Characterization of occult hepatitis B virus infection from blood donors in China

Prevalence and characteristics of occult hepatitis B virus (HBV) infection (OBI) of genotypes B and C prevalent in China have not been extensively explored. Characterization of OBI strains obtained from Chinese blood donors was based on clinical and serological analyses, follow-up testing, and sequence analyses. Twenty-eight samples from 165,371 HBV surface antigen (HBsAg)-negative plasmas were confirmed HBsAg negative and DNA positive(HBsAg(-)/DNA(+)), of which 22 were classified as OBIs and 6 as window period infections. The OBI incidence was 1:7,517 in blood donors, whose ages ranged between 20 and 45 years (median, 28 years). OBI donors had normal alanine aminotransferase (ALT) levels and low viral loads ranging between unquantifiable amounts and 178 IU/ml (median, 14 IU/ml). Sequences from 21 basic core promoter/precore (BCP/PC) regions, five whole genomes, and two additional pre-S/S regions from OBI strains were compared to genotypes B and C HBsAg(+) reference strains. Eighty-six percent (6/7) of OBI strains were genotype C. Deletions, insertions, stop codons, and substitutions were detected in 15/21 (71%) core regulatory elements of OBI strains. Critical mutations were found in the core proteins of 5/5 OBI strains in parallel with random substitutions in pre-S/S proteins from 6/7 (86%) OBI strains. Critical mutations in core regulatory elements and core proteins might affect OBI genotype B and C strain replication. That there were few S protein substitutions suggests a minor role of the host immune defenses in OBI occurrence.     

Full genome characterization of hepatitis B virus strains from blood donors in Iran

Iran is a low to medium endemic country for hepatitis B virus (HBV), depending on the region, where genotype D is dominant. Samples from 170 asymptomatic HBsAg‐positive blood donors were quantified and the median viral load was 6.7 × 10² IU/ml with 10.6% samples unquantifiable. Fifty complete genome sequences of these strains were characterized. Phylogenetic analysis identified 98% strains as subgenotype D1 and 2% as D2. Deduced serotypes were ayw2 (94%), ayw1 (4%), and adw (2%). The nucleotide diversity of the complete genome subgenotype D1 Iranian strains was limited (2.8%) and comparison with D1 strains from Egypt and Tunisia revealed little variation between strains from these three countries (range 1.9–2.8%). The molecular analysis of the individual genes revealed that the G1896A mutation was present in 86.2% of the strains and in 26 strains (29.9%) this mutation was accompanied by the G1899A mutation. The double mutations A1762T/G1764A and G1764T/C1766G were found in 20.7% and 24.1% of the strains, respectively. The pre‐C initiation codon was mutated in five strains (5.8%). One strain had a 2‐amino acid (aa) insertion at position s111 and another sP120Q substitution suggesting a vaccine escape mutant. J. Med. Virol. 83:948–952, 2011. © 2011 Wiley‐Liss, Inc.     

Surface antigen-negative hepatitis B virus infection in Dutch blood donors

Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of HBsAg-negative HBV infection in healthy persons and a comparison of the HBV genomes involved. The screening of 4.4 million Dutch blood donations identified 23 HBsAg-negative, HBV DNA-positive persons. Serological testing of the index donations, follow-up samples and archived earlier samples was performed to determine the nature of each HBV DNA-only case. Despite low viral loads HBV DNA could be sequenced in 14 out of 23 donors, allowing HBV genotyping and the analysis of mutations in the HBV surface gene. Four types of HBsAg-negative HBV infection were detected: infection in the early stage before occurrence of HBsAg; suppressed infection after vaccination; HBV genotype G infection with decreased HBsAg production; and chronic occult (HBsAg negative) HBV infection. In the donors with occult HBV genotype D infection the HBV surface gene showed multiple “escape” mutations in the HBsAg a-determinant and CTL epitopes, while in an occult genotype A case the surface gene showed no mutations. HBsAg-negative forms of HBV infection in healthy blood donors explain the ongoing transmission of HBV via blood transfusion, if donor screening is limited to HBsAg. The screening of blood donors for HBV DNA and HBV core antibodies seems to cover all stages and variants of HBV infection.     

Molecular epidemiological study of hepatitis B virus infection in two different ethnic populations from the Solomon Islands

The Solomon Islands is a multi-ethnic nation with a high rate of hepatitis B virus (HBV) infection. The prevalence relative to ethnicity was examined in relation to HBV infection, genotypes, and mutations. Asymptomatic populations (n = 564, 308 Melanesian and 118 Micronesian) from the Western Province were enrolled. Positive samples for Hepatitis B surface antigen (HBsAg) were examined for serological status, genotyping, viral load, and mutations of the basic core promoter (BCP) and pre-core (Pre-C) regions. The positive rate for HBsAg was 21.5%. The major Melanesian genotype was C (HBV/C), whereas the major Micronesian genotype was D (HBV/D). The prevalence of Hepatitis B e antigen (HBeAg) in serum was lower in carriers of HBV/D than of HBV/C. While the prevalence of the BCP mutation (T(1762)A(1764)) tended to be higher in HBV/C, that of the Pre-C mutation (T(1846)) was significantly higher in HBV/D (P < 0.0001). Genetic distance and phylogenetic analyses based on complete genome sequences were also carried out for two strains of HBV/C and two strains of HBV/D, and the findings were compared with those in the DDBJ/EMBL/GenBank database. The full-length sequence revealed that strains from the Solomon Islands were classified into subgenotype C3 (HBV/C3) and D4 (HBV/D4), and that the HBV/D strains were related closely to those from Papua New Guinea. HBV infection in the Solomon Islands is hyperendemic, and the genotype is ethnicity-specific. HBeAg appears to clear from the serum in young adulthood in HBV/D infection, which may be influenced by genotype-dependent features in relation to viral mutations.     

Molecular characterization of occult hepatitis B cases in Greek blood donors

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(?) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV‐1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(?) samples were tested further for HBV serological markers and HBV DNA was quantified by real‐time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti‐HBc only: 7 donors, anti‐HBc/anti‐HBs: 7 donors, anti‐HBc/anti‐HBe: 5 donors, anti‐HBc/anti‐HBs/anti‐HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively. J. Med. Virol. 81:815–825, 2009. © 2009 Wiley‐Liss, Inc.     

Diversity and origin of hepatitis B virus in Dutch blood donors

Two considerations led us to study the genetic diversity and origin of hepatitis B virus (HBV) in Dutch blood donors. Firstly, an HBV-infected Dutch blood donor was found negative by four assays used commonly for detection of HBV surface antigen (HBsAg). How variable is HBsAg among HBV infected blood donors? Secondly, the WHO recommends universal vaccination against HBV, but north-west European countries limit vaccination to groups at risk of HBV. This policy may reduce hepatitis B among low-risk, unvaccinated persons if HBV strains that infect low-risk persons stem from local at-risk groups. Studying the nucleotide sequence of the S-gene of HBV from 63 Dutch blood donors, considerable variation was found. The majority of the donor strains (52/63, 83%) appears closely related to local HBV isolates as present in intravenous drug users, immigrants, and homosexual men. The remaining 11 (17%) HBV strains belong to various non-Western genotypes. This implies that an indigenous Dutch HBV strain (heterosexually transmitted, not associated with intravenous drug abuse, or immigrants) does not exist, and it supports the policy in low endemic countries to limit vaccination to at-risk groups. On the other hand, it must be realised that, after 20 years of vaccination of at-risk groups, HBV still circulates in the at-risk groups and Dutch blood donors acquire the HBV strains involved. J. Med. Virol. 73:29–32, 2004. © 2004 Wiley-Liss, Inc.     

Antibodies against hepatitis B virus core antigen among blood donors]

The prevalence of antibodies to the hepatitis B virus (HBV) core antigen (anti-HBc) and the risk factors are evaluated in different blood donor groups. In 1998, on 12,456 first donations, 163 (1.31%) were positive with the two anti-HBc screening tests. Samples were from 69 women (42.3%) and 94 men (57.7%). Three subjects had no anti-HBs but were anti-HBc IgM negative. Forty (24.5%) donors were born in another country than France, and the majority (25 donors, 62.5%) were from North Africa. HBV vaccine had been previously given in 14 subjects (8.6%). Eight (4.9%) had hepatitis before the first donation. Trips in endemic areas (Africa and Asia) were taken by 26 subjects out of 76 donors with follow-up. Two (2.6%) had been previously transfused and six (7.9%) had contact with HBV-infected people. Among 78,033 repeat donations, 26 were positive with the two Anti-HBc screening tests (0.033%). Sixteen were negative after a second test and were probably false positive. Among the ten last donors, nine were Anti-HBs positive. One had anti-HBc IgM and had been recently infected by HBV. The prevalence of Anti-HBc in first-donation persons remains low. Trips in endemic areas and contact with an HBV-infected subject are the most frequent risk factors. Lastly, HBV seroconversion in repeat blood donors is a rare event. Anti-HBc screening in transfusion remains limited.     

Occult hepatitis B virus (HBV) infection in healthy blood donors

Blood transfusion is an important route of transmission of hepatitis B virus (HBV). Occult HBV infection can exist in the absence of HBsAg and can be detected by determining HBV DNA. To determine the occult HBV infection in healthy blood donors. One hundred adult healthy blood donors, negative for HBsAg, anti HCV, HIV-1 and other risk factors were screened for HBV DNA by PCR. All the healthy blood donors were negative for HBV DNA by PCR. Occult HBV infection does not occur in the healthy blood donors in the population studied.     

Molecular epidemiological study of hepatitis B virus among migrant workers from Cambodia,Laos, and Myanmar to Thailand

Although hepatitis B virus (HBV) infection is endemic in Southeast Asia, molecular epidemiological data on HBV circulating in some countries are limited. The aims of this study were to evaluate the seroprevalence of HBV and its genetic variability among migrant workers from Cambodia, Laos, and Myanmar in Thailand. Sera collected from 1,119 Cambodian, 787 Laotian, and 1,103 Myanmarese workers were tested for HBsAg. HBV DNA was amplified and the pre‐S/S region was sequenced for genotyping and genetic mutation analysis. HBsAg was detected in 282 (9.4%). The prevalence of HBsAg among migrant workers from Cambodia, Laos, and Myanmar was 10.8%, 6.9%, and 9.7%, respectively. Of 224 subjects positive for HBV DNA, 86% were classified as genotype C (99% were sub‐genotype C1) and 11.6% were genotype B (30.8%, 34.6%, and 30.8% were sub‐genotypes B2, B3, and B4, respectively). Various point mutations in the “a” determinant region were detected in approximately 18% of these samples, of which Ile126Ser/Asn was the most frequent variant. Sequencing analysis showed that 19.1% of samples had pre‐S mutations, with pre‐S2 deletion as the most common mutant (7.7%) followed by pre‐S2 start codon mutation (3.8%) and both pre‐S2 deletion and start codon mutation (3.3%). High prevalence of HBV infection (approximately 7–11%) was found among migrant workers from Cambodia, Laos, and Myanmar, which may reflect the current seroprevalence in their respective countries. The data also demonstrated that HBV sub‐genotype C1 was the predominant strain and various mutations of HBV occurring naturally were not uncommon among these populations. J. Med. Virol. 82:1341–1349, 2010. © 2010 Wiley‐Liss, Inc.     

低载量隐匿性乙型肝炎病毒检测和序列分析

目的对低载量隐匿性乙型肝炎病毒进行Q-PCR定量检测和BCP序列分析。方法采用诺华公司NAT方法筛查35332份献血者HBV DNA,有18例确认为HBsAg-/HBV DNA＋,48例为可疑HBV DNA＋血样,使用Q-PCR和Nested-PCR方法对48例可疑血样进行定量检测和BCP区域扩增和测序;并与野毒株BCP序列作比对分析。结果从48例可疑HBV DNA＋血样中成功应用Q-PCR定量检出HBV DNA阳性6例,均为隐匿性乙型肝炎（OBI）,并获得6例BCP序列,发现在TA富含区的变异相对较多,1例样品发生缺失变异,6例OBI血样共有14个变异位点在BCP区域。结论本方法能对可疑标本进行检测和定量,通过BCP区域的检测进行OBI的确认。     

献血人群隐匿性乙型肝炎病毒感染的流行病学和血清学研究

目的了解广州地区献血人群隐匿性乙型肝炎病毒感染（OBI）的流行病学和血清学情况。方法对广州地区199631例无偿献血者标本同时用ELISA法检测HBsAg、紫外-乳酸脱氢酶法检测ALT、核酸扩增技术（NAT）联合检测HBV/HCV/HIV及HBV单项鉴别试验,对HBsAg阴性HBV DNA阳性者进行随访,用荧光定量PCR检测病毒载量,用ELISA法检测乙肝两对半。结果 199631例标本中共检出104例HBsAg阴性HBV DNA阳性者,经随访有54例为OBI,OBI检出率为0.027%,年龄以46～55岁组检出率最高（P〈0.01）,外地身份证的献血者检出率高于广州市身份证者（P〈0.01）,OBI检出率与性别和献血次数无关（P〉0.05）。104例HBsAg阴性HBV DNA阳性的标本ALT均正常,病毒载量均〈1000IU/ml,平均值为162IU/ml。随访标本中,除6例ALT异常外其余均正常,54例OBI标本病毒载量均〈1000IU/ml,平均值为122IU/ml,乙肝两对半中抗-HBc阳性率明显高于其他项目（P〈0.01）。结论 HBsAg阴性献血者中存在OBI,有必要在献血者中开展核酸检测。     

沈阳地区乙型肝炎病毒基因型分子流行病学研究

目的 研究沈阳地区乙型肝炎病毒基因型分布和特点。方法 应用半巢式聚合酶链反应（PCR）扩增乙型肝炎病毒P基因，将PCR产物应用ABI377自动测序仪直接做核苷酸序列分析，并用DNA STAR软件进行种系发生分析及基因型鉴定。结果 HBV DNA标准株P基因片段可进行基因分型。在沈阳地区慢性HBV感染者中可检出基因型B、C和D，检出率分别为22％、50％和28％，基因型C分布与基因型B、D的差异有统计学意义，在慢性乙型肝炎患者和慢性HBV携带者间各基因型间分布比较中差异无统计学意义。结论 通过测定HBV DNAP基因序列片段可进行HBV DNA基因分型。沈阳地区乙型肝炎病毒基因型有基因型B、C和D型，其中基因型C为优势基因型。