乙型肝炎病毒基因聚合酶链反应分型方法的建立

根据乙型肝炎病毒（ＨＢＶ）的Ｓ区序列设计了３条引物：ＨＢＳ１、ＨＢＳ２和ＨＢＳ３，与ＨＢＳ１和ＨＢＳ２配对，经２次聚合酶链反应（ＰＣＲ）扩增。即可在检测有无ＨＢＶ－ＤＮＡ存在的同时对其基因型分类，可检出１０ａｇ的ＨＢＶ－ＤＮＡ，比免疫学方法更灵敏和特异。２５份不同亚型的标准血清中的绝大多数用Ｓ－ＰＣＲ和ＡＧＩＤ／ＲＰＨＡ的分型结果一致，Ｓ－ＰＣＲ的另一突出优越性的于能够对ＨＢｓＡｇ低滴度和阴性标本     

Hepatitis B surface antigen with an excess or deficiency in subtypic determinants in sera from asymptomatic carriers in Japan

Using the solid-phase enzyme immunoassay with monoclonal antibodies, hepatitis B surface antigen (HBsAg) was subtyped in sera from 5082 asymptomatic carriers who donated blood units at regional blood centers in Japan. Among them, 5004 sera contained HBsAg of a regular subtype, i.e., adw, adr, ayw or ayr, while 74 contained HBsAg with excessive subtypic determinants, such as adyw, adyr, adwr, aywr, or adywr. The presence of subtypic determinants on the selfsame particle was ascertained by sandwiching HBsAg between two monoclonal antibodies of distinct subtypic specificities. The remaining 4 sera contained HBsAg that possessed only one subtypic determinant, such as ad, ar or aw. HBsAg particles of atypical subtypes would have been given rise to by a point mutation in the S gene involving the codons regulating subtypic specificities.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.     

Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.     

Discrimination of hepatitis B virus (HBV) subtypes using monoclonal antibodies to the PreS1 and PreS2 domains of the viral envelope.

We report the production and characterization of murine anti-PreS2 and anti-PreS1 monoclonal antibodies (mAb) and demonstrate their utility in discriminating hepatitis B virus (HBV) subtypes. On the basis of Western blotting and reciprocal competition binding to HBV virions, at least five distinct epitopes have been identified in the PreS domain: two within the PreS1 region and three within the PreS2 region. All PreS2 mAb bind M protein (gp33 and gp36) but only one group binds strongly to M and L proteins (p39 and gp42). This group determinant was mapped to peptide residues 120-145. The second group bound to an endoglycosidase F-sensitive epitope which is defined by a mannose-rich glycan at ASN 123 in the PreS2 region. The third group was mapped to peptide residues 150-174 and was reactive with the M envelope proteins but not L or S proteins on Western blots. All PreS1 mAb bind L protein but not M protein on Western blots. Using these mAb, HBV subtype assays were developed allowing evaluation of the Paris (1975) HBsAg subtype panel members along with other HBsAg-positive specimens. All Paris subtype members (except ayw2 and ayw3) could be easily distinguished by differential PreS2 mAb reactivity. The Paris subtypes, adw2, adw4, and adr, could be classified as distinct groups by PreS2 and PreS1 mAb binding. Specimens from Hong Kong and the United States classified as adw2 in the S region fell into two groups based on PreS2 mAb binding: one having reactivity similar to Paris adw2 subtype and the other having identical reactivity to Paris ayw1 subtype. Furthermore, some specimens classified as adr in the S region gave similar reactivity to the Paris ayr subtype in the PreS2 and PreS1 regions. One complicating factor in this approach toward subtyping was the discovery that some HBsAg positive sera may contain factors which block PreS epitopes. Grouping of HBV subtypes by PreS1, PreS2, and S mAb reactivity may allow better correlation with groupings based on HBV DNA sequence homology.     

Characteristics of the X gene of hepatitis B virus

The 465-nucleotide sequence of the X gene from our cloned hepatitis B virus (HBV) DNA (subtype adw) was determined and compared to the same gene of 10 other published HBV sequences (3 of adw, 4 of adr, 2 of ayw, and 1 of ayr). We found (i) a total of 56 base differences among the 11 sequences (without counting the 27-base deletion in one adr) which resulted in 88% nucleotide homology, and (ii) 5 pairs of repeated sequence (3 direct repeats and 2 inverted repeats) that were highly conserved. Comparison of the protein amino acid sequences indicated that (i) there is 80% amino acid homology in total, and (ii) there are four highly conserved cysteine residues. In addition, the X gene of the adw subtype is more conserved than that of adr.     

Subtypes of HBsAG in Morocco: general distribution in HBsAG carriers in Casablanca in terms of their clinical state. Relations with the markers of the HBe system

HBs Ag subtypes ad and ay were determined by counter-electrophoresis (CEP) among 301 persons from Casablanca found HBs Ag positive by CEP: 147 were asymptomatic HBs Ag carriers and 154 were patients with acute or chronic hepatitis. w and r determinants were investigated among 82 from them only. HBe antigen and antibody (AB) were investigated by gel double immunodiffusion among 294 persons. Into the whole population, the prevalence of ad and ay subtypes was respectively 20 and 80%. This distribution, intermediate between those reported in West Europe and West Africa, was in agreement with data previously reported by others in the Maghreb, but differing from these ones in the distribution of w and r determinants. ayr subtype was found dominant (39%) and adw rare (2.4%), the prevalences of adr and ayw being intermediate (25-30%). Among asymptomatic HBs carriers and patients only adr and ayr distribution differed: in the formers, adr was dominant and ayr rare and, in contrast, ayr was frequent and adr rare among patients. Elsewhere, some findings were in agreement with previously reported data by others, such as the frequent association of ad to chronic liver disease or long-term HBs Ag carriage, ay being rather associated to short-term carriage, the frequency of HBe Ab in ad subtype and the presence of ay among almost all HBs positive haemodialysed patients.     

A synthetic peptide coded for by the pre-S2 region of hepatitis B virus for adding immunogenicity to small spherical particles made of the product of the S gene

Small spherical particles produced in the non-permissive phase of hepatitis B virus infection, when the viral genome is integrated into the chromosome of hosts, are rich in the product of the S gene, but poor in the product of the pre-S2 region. For the purpose of adding immunogenicity to spherical particles deficient in the pre-S2 region product, they were conjugated with a synthetic peptide of 19 amino acid residues. The peptide reproduced a hydrophilic area of the pre-S2 region product encoded by viral genomes of subtypes adr, ayw and ayr. The spherical particles supplemented with the pre-S2 peptide raised antibody to the pre-S2 region product in mice, in addition to antibody to the product of the S gene. Antibody to pre-S2 region product, prepared from sera of immunized mice by absorption with the S gene product, bound to spherical particles bearing pre-S2 region product, irrespective of adr, adw, ayw or ayr subtype, and agglutinated hepatitis B virions in immune electron microscopy. Based on the results obtained, the synthetic peptide may prove useful in adding protective efficacy to small spherical particles poor in pre-S2 region product.     

Different immunogenicity of H-2 Kb-restricted epitopes in natural variants of the hepatitis B surface antigen

The small hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV) has limited variability, but some serotypes and genotypes have been defined. Although no biological or pathogenetic differences could be traced to HBV serotypes, the clinical picture, response to treatment and long-term prognosis of HBV infection may vary with the HBV genotype, possibly due to differences in specific T cell recognition of HBV antigens from different genotypes. We analyzed murine CD8(+) T cell responses to two K(b)-restricted HBsAg epitopes primed by four different HBsAg variants using protein- and DNA-based vaccination protocols. The K(b)-binding S(208-215) epitope 1 is processed from exogenous but not endogenous HBsAg. Variants of epitope 1 differing at two positions within the epitope (ILSPFLPL in ayw/adr versus IVSPFIPL in adw2) efficiently primed cross-reactive CD8(+) T cell responses. In contrast, the exchange of an N-terminal flanking residue (S to N) completely eliminated the immunogenicity of epitope 1. The K(b)-binding S(190-197) epitope 2 is processed from endogenous but not exogenous HBsAg. A single-residue exchange within the epitope (VWLSVIWM in ayw/adr versus VWLSAIWM in adw2) completely eliminated the immunogenicity of epitope 2. Single, conservative residue exchanges can thus give rise to diverging CD8(+) T cell repertoires, suggesting an impressive complexity and flexibility of the CD8(+) T cell repertoire to antigen variants from viruses with limited diversity.     

Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.     

云南省乙型肝炎病毒基因型和血清型的流行状况分析

目的 了解云南省人群中流行的乙型肝炎病毒(Hepatitis B virus,HBV)基因型和血清型分布情况.方法 从参加健康体检的人群中筛查HBsAg阳性的血清标本,利用巢式PCR扩增HBV S基因片段,并对扩增产物进行序列测定.从GenBank中查获A～I基因型共27株HBV参考序列,构建HBV S基因系统发生树,以此确定其样本的基因型及血清型,并对结果进行统计分析.结果 从2216名体检人员血清标本中筛查出 39份HBsAg阳性的样本,HBV的S基因序列分型结果表明有4种基因型:C型占76.9%,B型占15.4%;D型占5.1%;I型占2.5%.血清分型结果为:adw2型占71.8%;adrq+型占17.9%;ayt型占10.3%.所有adw2血清型标本均为C基因型.HBsAg、HBeAg双阳性标本中75%为C基因型/adw2血清亚型.结论 云南省HBV感染人群中HBV基因型的分布以C型为主,血清型以adw2型为主.     

粤东客家地区乙型肝炎病毒S基因序列多态性分析

 目的：探讨广东客家地区乙型肝炎病毒（HBV）S基因序列的多态性，了解该地区S基因的流行病学特征。 方法： 运用基因扩增技术对40例来自广东客家地区的乙肝表面抗原阳性及HBVDNA阳性的HBV感染者S基因进行扩增，再对扩增产物进行DNA测序，将该地区S基因序列与标准序列对比分析。 结果： 40例样本中，adw型36例，adr型4例。36例adw型中，第131位天冬酰胺（Asn）均被苏氨酸（Thr）替代，其它位点也不同程度存在着氨基酸密码的差异：第127位脯氨酸（Pro）被Thr替代，第133位蛋氨酸(Met)被亮氨酸(Leu)或Thr替代, 第134位苯丙氨酸（Phe）被酪氨酸（Tyr）替代, 第143位Thr被Met替代，更多位点的遗传密码存在着简并性。而4例adr型的氨基酸序列高度同源。 结论： HBV亚型的分布具有明显的域性，广东客家人感染的HBV亚型主要为adw型，只有少数为adr型。与参照序列相比，adw型存在较明显的多态性。     

携带adr和adw两种亚型乙型肝炎病毒的病例

应用聚合酶链式反应（ＰＣＲ）扩增了一例疫苗保护失败的乙型肝炎病毒持续携带者及其母亲产前血清中ＨＢＶ Ｓ基因，并将其克隆入Ｍ１３ｍｐ１８中进行序列分析。发现患者及其母亲血清中均存在ａｄｒ和ａｄｗ两种亚型的ＨＢＶ ＤＮＡ。由其Ｓ基因推测出的氨基酸序列也分别与已报道的ａｄｒ和ａｄｗ两种亚型相符合。患者血清中ａｄｗ亚型存在第５８５位核苷酸Ａ变为Ｃ的点突变，导致Ｓ基因主蛋白第１４４位天门冬氨酸变为丙氨酸，并     

云南省乙型肝炎病毒基因型和血清型的流行状况分析

目的了解云南省人群中流行的乙型肝炎病毒（Hepatitis B virus,HBV）基因型和血清型分布情况。方法从参加健康体检的人群中筛查HBsAg阳性的血清标本,利用巢式PCR扩增HBVS基因片段,并对扩增产物进行序列测定,从GenBank中查获A～I基因型共27株HBV参考序列,构建HBVS基因系统发生树,以此确定其样本的基因型及血清型,并对结果进行统计分析。结果从2216名体检人员血清标本中筛查出39份HBsAg阳性的样本,HBV的s基因序列分型结果表明有4种基因型：c型占76．9％,B型占15．4％;D型占5．1％;I型占2．5％。血清分型结果为：adw2型占71．8％;adrq‘型占17．9％;ayr型占10．3％。所有adw2血清型标本均为c基因型。HBsAg、HBeAg双阳性标本中75％为c基因型／adw2血清亚型。结论云南省HBV感染人群中HBV基因型的分布以C型为主,血清型以adw2型为主。     

云南省乙型肝炎病毒基因型和血清型的流行状况分析

目的 了解云南省人群中流行的乙型肝炎病毒(Hepatitis B virus,HBV)基因型和血清型分布情况.方法 从参加健康体检的人群中筛查HBsAg阳性的血清标本,利用巢式PCR扩增HBV S基因片段,并对扩增产物进行序列测定.从GenBank中查获A～I基因型共27株HBV参考序列,构建HBV S基因系统发生树,以此确定其样本的基因型及血清型,并对结果进行统计分析.结果 从2216名体检人员血清标本中筛查出 39份HBsAg阳性的样本,HBV的S基因序列分型结果表明有4种基因型:C型占76.9%,B型占15.4%;D型占5.1%;I型占2.5%.血清分型结果为:adw2型占71.8%;adrq+型占17.9%;ayt型占10.3%.所有adw2血清型标本均为C基因型.HBsAg、HBeAg双阳性标本中75%为C基因型/adw2血清亚型.结论 云南省HBV感染人群中HBV基因型的分布以C型为主,血清型以adw2型为主.     

Impact of amino acid sequence variation of a determinant(s) on the antigenic properties of hepatitis B virus HBsAg

Impact of amino acid sequence variation on the antigenic properties of the surface hepatitis B virus antigen HBsAg was studied. Eleven recombinant HBsAg variants of wild (adr, ayw2, adw2, adw4, aywl, adw2) and vaccine escape (adw2 T126S, adw2 Q129L, adw2 Q129R, adw2 T143K, adw2 Q145R, aywl Q145A) were obtained. All the recombinant antigens were tested on a panel of 43 monoclonal antibodies (MAb) specific to different HBsAg determinants. Amino acid sequence variation of the a-determinant of HBsAg was shown to significantly affect its immunological responsiveness and antigenic properties. Amino acid substitution in different positions or in the same position, but for various amino acids may differently affect these properties.     

Molecular epidemiological study of hepatitis B virus in blood donors from five Chinese blood centers

Although the genetic variability of hepatitis B virus (HBV) in HBV-infected patients has been extensively studied, reports on genotypes, subtypes and mutations in the S region of HBV strains from Chinese blood donors are limited. In this study, 245 blood samples from HBsAg-positive blood donors were collected from five geographically diverse blood centers in China. The S region of HBV was amplified, and the HBV genotype and subtype were determined. The amino acid sequences of the S region were aligned, and mutations related to the failure of immunization and HBsAg detection were determined. Of the 245 samples, 228 (93?%) were genotyped successfully. We found that genotypes B, C, D and A accounted for 58.8?%, 21.9?%, 6.6?% and 3.95?% of the isolates, respectively. The distribution of HBV antigen subtypes was as follows: adw (67.6?%), adr (23.3?%) and ayw (8.7?%). Mutations were present in 39 (17.1?%) of 228 samples in the major hydrophilic region (MHR) of the S region. This study demonstrated that HBV genotype/subtype B/adw was the most frequent strain circulating in HBV-infected Chinese blood donors, followed by C/adr. The occurrence of MHR mutants in HBV-infected blood donors and the potential failure to detect some of them in collected units poses a threat to transfusion safety.     

Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIA.

Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies.     

Novel subgenotypes of hepatitis B virus genotypes C and D in Papua, Indonesia

Eight genotypes (A to H) and nine subtypes (adw2, adw4, ayw1, ayw2, ayw3, ayw4, adrq+, adrq-, and ayr) of hepatitis B virus (HBV) have been identified worldwide. They appear to be associated with geographical distribution, virological characteristics, and possibly clinical outcomes. We performed sequence analysis of part of the S gene and the entire precore/core gene of HBV isolates obtained from HBsAg-positive blood donors in Papua Province, Indonesia. Phylogenetic analysis of the S gene sequences revealed that 23 (85.2%) of the 27 HBV isolates tested belonged to genotype C (HBV/C) and 2 (7.4%) each to HBV/B and HBV/D. Interestingly, 19 (82.6%) of the 23 isolates of HBV/C clustered in a branch that was distinct from the previously reported subgenotypes C1 to C5 (HBV/C1 to HBV/C5). Similarly, two isolates of HBV/D clustered in a branch distinct from the reported subgenotypes HBV/D1 to HBV/D5. Phylogenetic analysis of the entire precore/core gene confirmed the consistent presence of the distinct branches in HBV/C and HBV/D. We therefore propose novel subgenotypes designated HBV/C6 and HBV/D6. The majority of HBV/C6 isolates in Papua had alanine at positions 159 and 177 (A159/A177) in the HBsAg. A159/A177 is different from the determinants for adrq+ (A159/V177), found throughout Asia, and adrq- (V159/A177), found in New Caledonia and Polynesia, possibly representing a unique antigenic group (provisionally referred to as adrq indeterminate). In conclusion, we have identified two novel HBV subgenotypes, HBV/C6 and HBV/D6, the first of which is the most prevalent subgenotype of HBV in Papua, Indonesia.