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.     

Another novel subgenotype of hepatitis B virus genotype C from papuans of Highland origin

Hepatitis B virus (HBV) genotypes and subtypes have been identified worldwide. As HBV genotypes/subtypes, the HBV subgenotypes seem to be associated with their geographical distribution and ethnic origin. A previous study showed the novel HBV subgenotype C6 based on the complete genome sequences of isolates in Papua, Indonesia. In the present study, further characterization of HBV in Jayapura (capital of Papua Province), particularly from native people of Papua originating from the highland (highland Papuans) and those from the lowland (lowland Papuans) were examined. Of 32 HBV isolates from both highland and lowland Papuan blood donors with HBsAg positive, part of the S gene and the core gene sequences were analyzed. Analyses of some isolates from highland Papuans were confirmed by the complete genome sequences. Most HBV isolates were classified into genotype C (78.1%), followed by genotype B (18.8%), and genotype D (3.1%). The subtype adr was predominant (71.9%), followed by adw2 (25.1%), and ayw2 (3.1%). As with previous findings, phylogenetic analyses revealed that most HBV isolates from Papuans, C/adr, belonged to subgenotype C6. Interestingly, some C/adr isolates from highland Papuans formed a distinct cluster from all reported subgenotypes of HBV/C, and they differed from HBV/C1-C10 by 4.2-7.2% over the complete genome. SimPlot analysis showed no evidence of recombination with HBV/C1-C10. The isolated life and closed social systems of highland Papuans, even though some have been moving to Jayapura, likely contribute to the formation of this unique cluster of infection with a novel subgenotype of HBV, named C11.     

Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India

Hepatitis B virus (HBV) has been classified into eight genotypes, and several subgenotypes, distinctly distributed geographically. The genotypes A and D were previously reported to be predominant in India. Recent studies indicated evidence of circulation of genotype C in Eastern part of India. With the aim to confirm the phylogenetic relation and molecular genetic characteristics of the HBV circulating in Kolkata, the most populous city in Eastern India, 11 strains were isolated and the complete genome sequences were analyzed. Phylogenetic analysis determined; three genotype C (adr-serotype) isolates closely related with C1 (Cs) subgenotype references from South East Asia, and three genotype A (adw2-serotype) isolates, related to Asia-variant references of subgenotype A1 (Aa). Whereas, five genotype D (ayw2, ayw3 serotype) isolates were highly divergent; one was related to subgenotype D1, two to subgenotype D3, and the remaining two clustered with a single genotype D isolate from Japan belonging to an unclassified subgenotype. Together, these two isolates differed from HBV D1-D4 subgenotypes by nucleotide differences ranging from 5.0 to 5.49%, probably indicating a new subgenotype, which we designate as D5. All serotype ayw3 of genotype D isolates had specific amino acid substitution Threonine at codon 118 and Methionine at codon 125 in antigenic determinant of surface gene that has not been reported previously in isolates from other parts of India. In conclusion; using the complete genome analyses this study has confirmed circulation of the genotype C in Eastern part of India and demonstrated considerable genotypic heterogeneity of the Indian genotype D.     

Molecular epidemiology of hepatitis B virus in Iran

Hepatitis B virus (HBV) infection is a major cause of liver disease worldwide. Eight genotypes and 24 subgenotypes of HBV have been identified. The aim of this study was to determine the distribution of HBV genotypes, subgenotypes and subtypes, and to understand HBV genetic variability in the HBV genome circulating in Iranian provinces. Two hundred and forty-nine sera from HBV-infected patients living in 25 provinces of Iran were collected (2004–2007). A part of the HBV S / pol and whole BCP / C genes were amplified, sequenced and then subjected to phylogenetic, recombination and genetic variability analysis. Results revealed genotype D of HBV in all samples and subgenotypes  D1 (98.52%), D2 (0.74%) and D3 (0.74%) among Iranian patients living in different provinces of Iran. Subtypes  ayw2 (94.4%), ayw1 (2.8%), ayw3 (2%) and ayw4 (0.4%) were deduced, on the basis of HBV small surface antigen (HBsAg) amino acid sequences. The mean percentage intra-genotypic distance of S plus core regions was 2.8%; the mean percentage inter-genotypic distance of this region between Iranian strains and genotype D isolates was 3.1%; and this rate for other genotypes was 5.2–11.4%. Various rates of point mutations have been found within different HBV genes, e.g. HBsAg (17.2%), precore-G1896A (59.5%) and Basal core promoter (BCP) double mutations (49.2%), whereas no recombination was found. In conclusion, these results indicate that the only genotype circulating in the provinces of Iran is genotype D. There exist high genetic variabilities in the S / pol and BCP / C regions among the Iranian HBV isolates.     

Incidence of genotype of hepatitis B subvirus and HBsAg subtypes in native people of northern and southeastern Siberia

Serological markers and DNA of hepatitis B virus (HBV) were detected in 487 blood samples of aboriginal people in the Alar district of the Irkutsk region (mostly Buryat) in 2005. HBsAg was found in 40 (8.2%) samples. HBV DNA was found in 24 out of 40 (60%) HBsAg-positive samples. HBV-positive DNA samples were found to contain nucleotide sequences of the Pre-S1, Pre-S2, and S regions of the HBV genome with a total length of 1146 n. 22 out of 24 (92%) isolates were found to belong to the D genotype, two belonged to the C genotype; eight (33.3%) belonged to the D3 subgenotype, six (25.0%) belonged to the D2 subgenotype, one (4.1%) belonged to the D1 subgenotype, and nine (37.5%) belonged to unidentified subgenotype. The incidence of the HBsAg subtype was determined to be ayw2 in 14 out of 24 (58.3%) isolates and ayw3 in seven (29.2%) isolates; the subtype was not identified for one (4.1%) isolate. In two C-genotype isolates, the subtypes were identified as adw2 and adrq+. A comparative analysis of the results of this work and those obtained previously for native people of Yamalo-Nenets Autonomous District (YaNAD, mostly Khanty and Komi) demonstrated significant differences in the incidence in YaNAD of HBsAg (3.2% isolates, p > 0.999), subgenotype D2 (62% isolates, p > 0.95), and subtype ayw3 (70.6% isolates; p > 0.95). The variability in the incidence of two variants in two groups of native Siberian peoples is the evidence of different infection sources in these populations.     

Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.     

A nationwide molecular epidemiological study on hepatitis B virus in Indonesia: identification of two novel subgenotypes,B8 and C7

Upon phylogenetic analysis of a partial S gene sequence [396 nucleotides (nt)], 928 hepatitis B virus (HBV) strains obtained from 899 viremic subjects in 28 major cities on 15 islands of Indonesia in 1989–2007 segregated into four HBV genotypes. Genotype B was predominant (66%), followed by genotype C (26%), genotype D (7%), and genotype A (0.8%). Comparative and phylogenetic analyses of the 396-nt S gene sequence of 928 HBV isolates and whole genomic sequences of 25 selected HBV isolates revealed a total of 14 subgenotypes within genotypes A–D: two (A1 and A2) in genotype A (HBV/A), five (B2, B3, B5, B7, and a novel subgenotype, tentatively designated B8) in HBV/B, five (C1, C2, C5, C6, and another novel subgenotype, C7) in HBV/C, and two (D1 and D3) in HBV/D. The distribution of HBV genotypes/subgenotypes, including B8 and C7, seems to be associated with ethnological origins in Indonesia. The nucleotide sequence data reported in this study have been assigned DDBJ/EMBL/GenBank accession numbers AP011084–AP011108 for 25 entire HBV genomes and AB466339–AB467266 for 928 partial HBV sequences.     

Prevalence of hepatitis B virus MHR mutations and their correlation with genotypes and antiviral therapy in chronically infected patients in Serbia

Understanding the prevalence and diversity of HBsAg variants in a population is fundamental to assay design and planning vaccination programs. It has been shown that mutations within the S gene, caused by selection or natural variation, can lead to false‐negative results in assays for HBsAg, or have clinical implications, such as evading anti‐HBV immunoglobulin therapy or vaccine‐induced immunity. The region of HBsAg where most of these mutations occur is known as the major hydrophilic region (MHR). The aim of this study was to determine the prevalence and mutational patterns of MHR mutations in patients with chronic hepatitis B, and their correlation with patient characteristics, viral factors and antiviral therapy. The study comprised 164 plasma samples from patients with chronic hepatitis B, of which, 34.8% were on long‐term lamivudine monotherapy. Direct sequencing of part of the S/pol gene was used for identification of HBsAg mutations, HBV genotypes, subgenotypes and HBsAg subtypes. The overall frequency of MHR mutations was 22.6%, but it varied significantly between untreated and treated patients (16.8% vs. 33.3%). The most frequent substitution was at position 120 (9.1%) whereas the most common vaccine‐escape position, 145, was affected in 1.8% of isolates. The presence of MHR mutations was correlated with genotype D, subgenotype D3, and ayw2/ayw3 HBsAg subtypes and to older age (>40 years). It is concluded that natural viral variability present in a geographical region, duration of infection, and antiviral therapy are among the major factors associated with the occurrence of MHR mutations. J. Med. Virol. 82: 1160–1167, 2010. © 2010 Wiley‐Liss, Inc.     

Hepatitis B virus subgenotype A1, occurrence of subgenotype D4, and S gene mutations among voluntary blood donors in Kenya

Kenya is one of the high endemic zones for hepatitis B virus (HBV) infection. The consensuses on prevalence of the HBV genotypes and the existence of their variants have not been fully established in Kenya. Hence, there is a need to further monitor the diversity of HBV. This study aimed to extend the current molecular and epidemiological information about the geographical distribution of HBV genotypes and subgenotypes, as well as to describe the hepatitis B surface antigen (HBsAg) variants circulating in different Regional Blood Transfusion Centres of Kenya. A total of 32 HBsAg positive blood units from five different blood transfusion centers in Kenya were used in the study. The HBV DNA preS/S-gene was amplified and sequenced. Alignments of S gene were applied using reference sequence from GeneBank. Phylogenetic analysis was performed using the MEGAv4.0 software with the neighbor-joining and maximum composite likelihood methods. Twenty-one plasma samples (65.6 %) were DNA positive and were successfully sequenced. Eighteen out of the twenty-one isolates (85.7 %) belonged to subgenotype A1 Afro-Asian: six were from Nairobi, four from Kisumu, two from Embu, and three each from Eldoret and Mombasa. The other three strains (14.3 %, 3/21) belonged to subgenotype D4 from Mombasa. The HBsAg mutations were detected in nine isolates (42.9 %, 9/21). The HBV/A1 and HBV/D4 are dominant among blood donors in Kenya. This demonstrates that continuous monitoring of the HBV diversity would help reveal circulating genotypes and subgenotypes as well as mutants of clinical significance in Kenya.     

Prevalent HBV genotypes and subtypes in a South Indian population.

BACKGROUND: Genotypes or genetic subtypes describe genetically related strains and have been described for viruses belonging to several different families. The eight major genotypes of hepatitis B virus (HBV) have distinct geographic distribution. Recent studies suggest possible pathogenic and therapeutic differences among HBV genotypes. OBJECTIVES: To evaluate the HBV genotypes of 85 samples by RFLP analysis and sequence the desired region to look for variations and identify the subtypes of the surface region. STUDY DESIGN: We studied 85 patients with HBV in order to identify the most prevalent genotype and subtype. Patients with HBV-related liver disease attending the Department of Gastroenterology at Owaisi Hospital and Research Centre were studied. RESULTS AND CONCLUSIONS: Genotype D1 was most prevalent. Genotyping was carried out by RFLP analysis and confirmed by sequencing. Nucleotide sequences showed significant homology (96-97%) with the other genotypes that have been reported. Subtype ayw was the most prevalent subtype within the surface region. Construction of a phylogenetic tree incorporating these isolates and other published HBV sequences showed that the isolates are derived from the same evolutionary tree. The study adds to our understanding of the genetic diversity of HBV and the geographical distribution of its subtypes, and will be useful for reconstructing the evolutionary history of HBV.     

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.     

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

Co–circulation of different genotype,sub-genotype and serotypes of hepatitis B virus (HBV) and its epidemiology in Assam: A north-eastern state of India

PurposeHepatitis B virus (HBV) infection is a global health problem. HBV has different genotypes and subgenotypes with geographical distinctiveness. Aims: To study the molecular epidemiology and distribution pattern of HBV in Assam; a distinct state of India that may have different genotypic divergence. Settings and design: Patients attending a tertiary care hospital susceptive of Hepatitis B were included, irrespective of age and sex in different agro-climatic zones of Assam.MethodsSamples positive for Hepatitis B surface antigen test and COBAS®TaqMan® HBV tests were further confirmed by PCR and sequencing followed by phylogenetic analysis.Statistical analysis used: Chi-square test was used to determine whether there was a significant difference among the results in this study.Results: An increase trend of HBV positive cases has been observed in the state. The incidence in female was lower than that of male and age group 26–35 years was most vulnerable. Genotype D, subgenotype D2 and serotype ayw3 were predominant genotype, subgenotype and serotype. The prevalence of subgenotype C3 was a new finding. Phylogenetic analysis showed that the genotypes of HBV prevailing in the state have close relationship with neighboring countries of India which may be due to increased cross border migration of populationConclusionsThis comprehensive study of HBV in Assam described the distribution of HBV genotypes and subgenotypes and serotypes in different agro-climatic zones of Assam. These findings will help to formulate the roadmap for prevention and control of the disease as well as targeted therapy of HBV in this State.     

A new isolate of hepatitis B virus from the Philippines possibly representing a new subgenotype C6

Hepatitis B virus (HBV) genotypes and subgenotypes show distinct geographical prevalence. A genotyping analysis of 28 samples from asymptomatic HBV carriers from the Philippines gave a distribution of HBV genotypes as expected from a previous study: 54% B (15/28), C5 18% (5/28), 14% D (4/28), 7% A1 (2/28). In addition, 7% (2/28) of the samples showed a double infection with genotypes B and D. One of the isolates sequenced completely, ph105, did not group into one of the known subgenotypes after phylogenetic analysis. Ph105 formed a separate clade in genotype C. With a genome length of 3,215 nt. and a serological subtype adr, ph105 exhibited the main features of most genotype C strains. However, ph105 differed by 4.1–7.2% from HBV subgenotypes C1 to C5 when comparing the nucleotide sequence of whole genomes. With only 4.1% difference ph105 was most closely related to subgenotype C2. SimPlot analysis gave no indication for recombination with known HBV genotypes. Ph105 fulfils all criteria for a new subgenotype C6. J. Med. Virol. 81:983–987, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of hepatitis B virus genome variability in Iranian patients with chronic infection, a nationwide study

Hepatitis B virus (HBV) isolates from Iranian patients around the country were characterized. Eighty-one complete genomes from HBV isolates were sequenced and analyzed. The studied population was grouped into three categories including inactive carriers, patients with chronic hepatitis, and patients with liver cirrhosis. Molecular and phylogenetic analyses revealed that Iranian patients were infected with HBV genotype D and subgenotype D1. The most common subtype was ayw2, followed by ayw3 and ayw4. Several deletions and insertions that had no correlation with disease outcome were observed in the HBV genomes. The most frequent mutation in the major hydrophilic region (MHR) of HBV surface antigen (HBsAg) was sP120S. Almost half of the patients studied carried precore (PC) mutant variants and one-third of the studied population was infected with variants carrying basal core promoter (BCP) mutations. PC and BCP mutations were observed in older patients, especially in those with chronic liver disease. Sixty-seven patients (82.7%) were HBeAg negative, and the prevalence of precore mutant isolates (G1896A) was higher in this group than in HBeAg-positive patients. Lamivudine drug resistance mutations were detected after 1 year of treatment in about 30% of lamivudine-treated patients. In conclusion, these results demonstrate that HBV subgenotype D1 is the only subgenotype circulating in Iran, and there is no evidence of any exotic genotype in the region. The HBV PC (G1896A) mutation may play an important role in the clinical outcome of the disease by increasing the risk of progressive liver disease among Iranian patients infected with HBV.     

Identification and characterization of novel hepatitis B virus subgenotype C10 in Nusa Tenggara, Indonesia

Six novel subgenotypes (B7, B8, C6, C8, C9, and D6) within three hepatitis B virus (HBV) genotypes (B–D) were recently identified in Indonesia. To further characterize HBV in this country, 18 HBV-viremic samples obtained from blood donors in Nusa Tenggara, Indonesia, were subjected to phylogenetic analysis of an 1.6-kb partial or full-length sequence. Thirteen HBV isolates were classified into genotype B with four distinct subgenotypes [B3 (n = 2), B5 (n = 1), B7 (n = 4), and B8 (n = 6)], followed by 4 HBV isolates of genotype C (HBV/C); the remaining one isolate was of D (D1). As for the four HBV/C isolates, one isolate segregated into subgenotype C1, and two into C2. The remaining HBV/C isolate [C0901177(NT3)] differed from reported HBV/C isolates (C1–C9) by 4.6–7.7% over the entire genome and did not show evidence of recombination with any of the known HBV genotypes/subgenotypes, justifying its conclusive assignment into a novel subgenotype (C10) within genotype C.     

Hepatitis B virus subgenotype C2 is the most prevalent subgenotype in northeast China

The geographical distribution of hepatitis B virus (HBV) subgenotypes and their clinical implications in patients with acute and chronic hepatitis B in the Heilung-kiang province of northeast China were investigated. Nested PCR and multiplex PCR were performed with genotype-specific primers and with subgenotype-specific primers to identify genotypes and subgenotypes from serum samples of 412 HBV infections including 69 with acute self-limited hepatitis (ASH) and 343 with chronic hepatitis (CH). A total of 361 samples were genotyped and 304 were further subgenotyped. The most common HBV genotype was C (93.63%, 338/361), with subgenotype group C2 (83.73%, 283/338) predominating. Genotype B was also found and subgenotype B2 predominated within this genotype. Out of 69 infected patients with ASH, 48 were identified as genotype C and all belonged to subgenotype C2. Of 343 infected patients with CH, 313 were genotyped and 256 were subgenotyped; amongst these, C2 (91.80%, 235/256), B2 (7.42%, 19/256) and mixed subgenotypes B2 and C2 (0.78%, 2/256) were found. In HBV subgenotype C2 infections, ASH had a higher ratio of women than CH patients. These results show that HBV subgenotypes C2 and B2 were found in Heilung-kiang province of northeast China. In ASH and CH groups, the distributions of subgenotypes were coincident with C2, the predominant subgenotype. Analysis of the association between subgenotype and the outcomes of HBV infection was inconclusive in our study.     

Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland

Hepatitis B virus (HBV) infection is one of the major global epidemiological problems. The aim of our study was to determine the distribution of HBV genotypes in Poland since the data concerning the spread of HBV viruses in the central-eastern region of Europe is still very limited. HBV DNA was extracted from 58 serum samples. To quantify the level of HBV DNA the Roche Amplicor HBV Monitor Assay was used. To genotype and assign HBV subtypes DNA sequencing methods were performed. The HBV virus from 43 serum samples from hepatitis B infected patients was genotype A (74.1%), 12 cases had genotype D (20.7%), and 3 had the rare in Europe genotype F (5.2%). Prediction of HBV serological subtypes based on HBsAg sequencing showed almost 100% occurrence of subtype adw2 in the group of genotype A samples, three different subtypes in genotype D (ayw2, ayw3, and ayw4), and equal distribution of subtype adw4q- in all 3 cases of genotype F, also the most prevalent subtype in the Amerindians. Our results coincide with the general European HBV prevalence. However, HBV genotype F, which is not a common genotype in European countries, was detected and so was relatively high occurrence of genotype D, which may reflect historical and ethnical migration events in Poland in the past.     

Novel quasi-subgenotype D2 of hepatitis B virus identified in Taiwanese aborigines

The hepatitis B virus (HBV) genomes in Taiwanese aborigines, whose ancestors have lived in Taiwan for over 10,000 years, have not been characterized. In order to characterize of HBV in this special population, serum samples were obtained from serologically HBsAg-positive 27 Taiwanese aborigines. The pre-S1/S2 region and the full-length 3.2 kb of the HBV genome were amplified by PCR. Obtained amplicons were sequenced and confirmed the HBV genotypes by phylogenetic analysis. By phylogenetic analysis of the sequence of pre-S1/pre-S2 region, HBV/B2 (21/27: 78 %) was the most prevalent followed by genotype D (6/27: 22 %). Two strains of HBV/B2, each having 3,215 bp genomes, had recombination with genotype C in the pre-C/C gene which is characteristic of subgenotype B2 circulating in Southeast Asia. Interestingly, six strains of genotype D formed a distinct cluster between subgenotypes D1 and D2 suggesting a novel group of HBV. A similar finding could also be confirmed based on the entire 3,182 bp genome from four strains of HBV/D. This new cluster was supported by a branch with 99 % bootstrap value and 3.4–5.8 % nucleotide divergence over the entire genome from other known subgenotypes D1 to D9. Four strains of the new D subgenotype showed serotype ayw2, but had unique amino acid sequences consisting of N115 in the preS/S gene; P41 in the X gene; S239, K/E295, V567, and P708 in the P gene, respectively. From the above results, we provisionally proposed to designate it as novel quasi-subgenotype D2 identified in Taiwanese aborigines.     

Five subgenotypes of hepatitis B virus genotype B with distinct geographic and virological characteristics

Several hepatitis B virus (HBV) subgenotypes, HBV/A1, A2, Bj and Ba, have been reported with respect to clinical differences among patients infected with these subgenotypes. The population genetics and phylogeography of HBV were investigated based on the complete genome sequences of 484 isolates with 108 from our chronic hepatitis B patients and the remaining from the GenBank database. Besides genotypes A-H (HBV/A-H), five subgenotypes were identified among 169 HBV/B isolates by phylogenetic analysis and nucleotide divergence. There were 27 isolates of subgenotype B(1) (HBV/B(1)) restricted to Japan, 104 isolates of HBV/B(2) with the widest distribution in most Asian countries, 4 isolates of HBV/B(3) restricted to Indonesia, 32 isolates of HBV/B(4) restricted to Vietnam, and 7 isolates of HBV/B(5) restricted to Philippines. HBV/B(2)-B(5) isolates carried a recombination with HBV/C over the precore and core genes. In addition to the characteristics of HBV/B(1)-B(5) at some cis-acting elements, the precore stop-codon mutant (G1896A) was significantly different among HBV/B(1), HBV/B(2), and HBV/B(4) (70.3%, 31.7%, 53.0%, P=0.001), while no such mutation was found in HBV/B(3) and B(5). Among characteristics of the HBV/B(1)-B(5) amino acid sequences, serotype adw (K(122)) was exclusive among HBV/B(1), HBV/B(2), and HB V/B(3) isolates, while serotype ayw (R(122)) was among the HBV/B(4) and HBV/B(5) isolates. Furthermore, distinct variations of T cell and B cell recognition epitopes within surface and core proteins were also found among these subgenotypes. In conclusion, subgenotypes HBV/B(1)-B(5) exhibited distinct geographical distributions, virologic characteristics, and probable clinical implications.