Subgenotype C2 Research Articles

Prevalence of genotypes and subtypes of hepatitis B viruses in Bangladeshi population.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergences in the entire genome of >8 %, HBV genomes have been classified into ten genotypes designated as A to J. The aim of this study was to determine HBV genotypes and subtype in samples of HBV infected patients in Bangladesh. The sera samples were collected from chronically infected HBV patients. At first the DNA positive HBV samples were screened by EIA in our laboratory and the 1063 bp region of surface gene was amplified, sequenced and genotyped by sequence analysis. The same sequences were also used for subtypes and mutational analyses. After that, genotyping was also carried out by nested PCR using genotype specific primers in the same region of HBV surface gene. A total of 39 samples were sequencing to find out the genotypes and subtypes. It was found that the prevalent genotype was genotype C (subgenotype C1) which accounted for 48.7 %. The other genotypes found were genotype A (23.1 %) and genotype D (28.2 %). Predominant subtypes in Bangladesh were adr (41 %) followed by subtype adw2 (28.2 %), ayw3 (25.6 %), and others. Additionally, genotyping was also done by nested PCR using type-specific primers. In this method, out of 17 samples 6 were found to be genotype C, followed by genotype D (4 of 17) and genotype A (3 of 17). In PCR-based genotyping system we also observed the mix genotypes; 3 samples contained both genotype A and D, and 2 samples contained both C and D. The genetic diversity of HBV and distribution of its genotypes and subtypes amongst Bangladeshi population were done in this study, which will help us to provide information regarding circulating genotypes in this region and also help physicians to prescribe proper antiviral/interferon therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1840-2) contains supplementary material, which is available to authorized users.

Occult HBV Infection May Be Transmitted through Close Contact and Manifest as an Overt Infection.

The importance of transmission of occult HBV infection (OBI) via transfusion, organ transplantation and hemodialysis has been widely recognized. However, data regarding the transmission of OBI through close contact remain limited. In this study, serum samples were obtained from a child and his parents. The child had received the standard vaccination regimen at birth and produced protective antibody. Sera were tested for HBV serological markers. Nested PCR assays were used to detect HBV DNA and the amplicons were cloned and their sequences subjected to phylogenetic analysis. The results showed that both parents had occult infections while the child had an overt infection. Twelve, eleven and nine clones, from the father, mother and son, respectively, were sequenced. Serotypes adrq+, ayw1, ayw and ayr were found in the father and ayw1, adw2 and adwq+ in the mother; adrq+ was the only serotype in son. Genotype B, subgenotype C2 and a recombinant were identified in the father and genotype B, subgenotype C5 and three recombinants were found in the mother. Subgenotype C2 was the only genotype identified in the child. A phylogenetic tree showed that all of the child’s sequences and most of the father’s sequences clustered together. However, none of mother’s sequences clustered with those of the child. The surface gene from the child and his father had the same amino acid substitution pattern (T118K, T123N and G145A). We concluded that the father was the source of the son’s HBV infection, suggesting that occult HBV infection may be transmitted through close contact and manifest as an overt infection.

A molecular epidemiological study of the hepatitis B virus in Thailand after 22 years of universal immunization.

Hepatitis B virus (HBV) infection affects an estimated two billion people worldwide. Since 1992, Thailand implemented universal HBV vaccination as part of the expanded program on immunization (EPI) for newborns. This study aims to compare genotypes and characterize HBV by assessing pre-S/S and basic core promoter (BCP)/precore (PC) mutations in populations born before and after EPI implementation. A nationwide serosurvey conducted in 2014 assessed the impact of universal HBV vaccination in Thailand. Two cohort groups were established based on whether they were born before or after 1992. HBV DNA was amplified from HBsAg positive samples by PCR and sequenced. HBV genotypes, pre-S/S regions, and BCP/PC mutations were characterized. From a total of 5,964 subjects, there were 2,805 (47.0%) and 3,159 (53.0%) individuals who were born before and after EPI implementation, respectively. The overall prevalence of HBsAg was 2.2%. The prevalence of HBsAg was significantly higher in the before EPI group (4.3%) than in the after EPI group (0.3%) (P < 0.001). HBV DNA was detected in 119 samples; 111 HBV-positive samples (93%) were genotype C (subgenotype C1). The "a" determinant mutation was only detected in the "before EPI" group. Twenty-two years after implementation of the EPI program, the HBV carrier rate is significantly reduced. The most prevalent genotype for the remaining HBV was C1. The "vaccine escape" mutant, especially the "a" determinant, was not detected after the launch of the EPI program, and the current HBV vaccine remains highly effective.

Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 107 TCID50/mL 10 days after infection when using an MOI of 10−4. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

Relationship between HBV genotypes/sub-genotypes of hepatitis B virus and antiviral therapy

Objective To investigate the relationship between the genotype/sub-genotype and antiviral treatment. Methods The clinical data of 159 patients with chronic HBV infections were collected, who were treated by adefovir dipivoxil(10mg/d)or entecavir(0.5mg/d)according to the disease's conditions. The genotypes and subtypes of hepatitis B virus were determined by Nested polymerase chain reaction(nt PCR). The levels of serum HBVDNA replication, ALT levels and HBV serologic markers were detected pre or post-treatment 24 weeks,48 weeks. Observed the relationship between the HBV genotypes/sub-genotypes and the antiviral efficacy of adefovir dipivoxil or entecavir treatment. Results After 24 weeks of adefovir dipivoxil therapy,ALT normalization rate of subgenotype Ba and subgenotype C2 was 66.7%vs 66.2%(χ2=0.74,P>0.05),HBeAg negative conversion rate of two subgenotypes was 27.3%vs 23.1%(χ2=0.10,P>0.05),HBVDNA negative conversion rate of two sub genotype Ba and subgenotype C2 was 33.3%vs 30.9%(χ2=0.03,P>0.05),respectively.After treatment for 48 weeks,ALT normalization rates of subgenotype Ba and subgenotype C2 were 83.3%vs 82.4%(χ2=0.01,P>0.05),HBeAg negative conversion rates of two subgenotypes were 45.5%vs 34.4%(χ2=0.49,P>0.05),HBVDNA negative conversion rates of two subgenotypes were 58.3%vs 48.5%(χ2=0.39,P>0.05).After 24 weeks of entecavir therapy,ALT normalization rates of subgenotype Ba and subgenotype C2 were 71.4%vs 69.6%(χ2=0.02,P>0.05),HBeAg negative conversion rates of two subgenotypes were 33.3%vs 30.8%(χ2=0.03,P>0.05),HBVDNA negative conversion rates of two subgenotypes were 42.9%vs 39.3%(χ2=0.06,P>0.05),respectively. After treatment for 48 weeks,ALT normali zation rates of subgenotype Ba and subgenotype C2 were 85.7%vs 83.9% (χ2=0.03,P>0.05),HBeAg negative conversion rates of two subgenotypes were 50.0%vs 44.9%(χ2=0.10, P>0.05),HBVDNA negative conversion rates of two subgenotypes were 71.4%vs 67.8%(χ2=0.07,P>0.05). Conclusion The study showed that genotype C(C2)is a predominant HBV genotype among people with chronic HBV infections in Shanxi province.HBV subgenotypes Ba and C2 have no significant difference in virologic, biochemical, and immunologic response to ADV or ETV. Key words: Chronic Hepatitis B; Genotype; Adefovir Dipivoxil; Entecavir

Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade.

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.

The prevalence of mutations in the major hydrophilic region of the surface antigen of hepatitis B virus varies with subgenotype

Mutations in the major hydrophilic region (MHR) of the surface antigen of hepatitis B virus (HBV) may result in vaccine escape, failure of immunotherapy and antiviral resistance. These mutants may be transmitted and constitute a public health threat. We aimed to determine the prevalence of MHR mutations of HBV in areas of high endemicity in Guangxi, China. HBV surface gene was analysed from 278 HBsAg-positive asymptomatic individuals recruited from Guangxi using cluster sampling. Three genotypes, B, C and I, were identified. The overall prevalence of MHR mutations is 17·6%. The prevalence of MHR mutations in genotype B (15·1%) is not significantly different from that in genotype C (16·4%). However, the prevalence in subgenotype C5 (31·1%) is significantly higher than in subgenotype C2 (13·0%) (χ 2 = 6·997, P < 0·05). The prevalence of escape mutations and overlapping polymerase substitutions in subgenotype C5 is significantly higher than in subgenotypes B2 and C2. In total, 7·9% of MHR mutants are escape mutations and 72·1% of MHR mutations produced amino-acid changes in the overlapping polymerase, including resistance mutations to entecavir. Our results suggest that the prevalence of MHR mutations varies with subgenotype. The prevalence of escape mutations and polymerase mutations may be associated with subgenotype.

Association of human leukocyte antigen haplotypes with clearance and persistence of hepatitis B virus infection in northeastern China

This study investigated clinical implications of human leukocyte antigen (HLA) I and II haplotypes, in combination with HBV sub-genotype C2, in hepatitis B virus (HBV) infections in northeastern China. Here, HLA haplotypes of 230 HBV-infected patients were compared to 210 healthy, unrelated Han individuals. Of the 230 HBV-infected patients, 54 had acute self-limited hepatitis (ASH) with sub-genotype C2 (ASH-C2), 144 had chronic hepatitis (CH) with sub-genotypes C2 and B2 (CH-C2 and CH-B2), and 32 spontaneously recovered without sub-genotype results. All groups underwent HLA typing and haplotype analysis. The results revealed that A*02-DRB1*12 and A*02-B*15-DRB1*09 carriers were susceptible to HBV infection. A*02-B*15-DRB1*09 is probably associated with acute onset and viral clearance and A*02-DRB1*12, with viral persistence. In HBV infections, B*40-DRB1*12 was associated with HBV persistence, whereas B*46-DRB1*09, A*24-DRB1*14, and B*15-DRB1*04 carriers easily recovered from the disease. By contrast, when infected with the HBV-C2 sub-genotype, A*24-DRB1*14, B*15-DRB1*04, A*02-B*15, A*02-DRB1*15, and A*02-B*15-DRB1*09 carriers displayed an acute clinical course before recovery. This study reveals a relationship between HLA haplotypes and HBV pathogenesis, thereby providing potential therapeutic targets to treat HBV infection.

The prevalence of HBsAg subtypes and HBV genotypes in native population groups of Siberia

The prevalence of HBsAg and its subtypes and hepatitis B virus (HBV) genotypes in native population groups of five Siberian regions—the Republic of Altai, Kemerovo oblast, Irkutsk oblast, Yamalo-Nenet autonomous oblast (the YaNAO), and Kransnoyarsk krai (a total of 5657 samples)—was studied. Statistically significant differences were found in these groups for the studied HBV markers and types. HBsAg was the most prevalent in Altaians in Republic of Altai (13.4% of samples); Dolgans and Nganasans in Krasnoyarsk krai (13.2%); Teleuts in Kemerovo oblast (10.2%); and Buryats in Irkutsk oblast (5.4–8.2%). HBsAg prevalence was minor in Khants, Komi, Nenets and Selkups in YaNAO (0.7–1.7%). The study of the partial HBsAg-gene sequences of 143 HBV isolates from different groups of the five Siberian regions revealed that 130 (90.9%) belonged to genotype D (subtypes ayw2 and ayw3), 10 (7%) to genotype C (subtype adrq+), and 3 (2.1%) to genotype A (subtype adw2). Of the 130 genotype D isolates, 120 belonged to subgenotypes D1 (34.3%), D2 (22.4%), and D3 (27.3%), while the genotype was not determined in 10 (7%) isolates. Subgenotype D1 (HBsAg subtype ayw2) was prevalent among Kazakhs in the Republic of Altai (85.7% of the strains), Teleuts in Kemerovo oblast (60.0%), Russians in Irkutsk oblast (87.5%), and Dolgans and Nganasans in Krasnoyarsk krai (68.8%). Subgenotype D2 (subtype ayw3) was prevalent among Khants and Komi (75%) and Nentsi (57.1%) from the YaNAO. Subgenotype D3 (subtype ayw2) was prevalent in Altaians in the Republic of Altai (76.2%) and Buryats from Irkutsk oblast (50%). Significant prevalence of genotype C (subgenotype C1, 18.8%) was first found in the Far North of Siberia (Taimyr Peninsula, Krasnoyarsk krai). On the basis of the obtained data, we suggested that there exist several epidemiologically different sources of HBV expansion in native Siberian population groups.

Establishment of reference sequences of hepatitis B virus genotype C subgenotypes.

Hepatitis B virus genotype C (HBV/C) has the largest number of subgenotypes (C1-C16) that vary with geography and isolates. HBV/C prevails in Southeast Asia (C1, C5-C16), East Asia (C2), Oceania (C3), and Australia (C4). Suitable reference strains for different subgenotypes could greatly facilitate research into HBV/C, but unfortunately they are scarce. We retrieved 974 HBV/C full-length sequences from the GenBank database and subgenotyped them by phylogenetic analysis. Reference sequences of each subgenotype from different locations were established with the most frequent nucleotide present at each position of the isolates that belonged to the same subgenotype. The reference sequences of subgenotypes C1, C2, C5, and C6 have been constructed and deposited in GenBank (KM999990-KM999993). The homology between the reference sequences and almost all the isolates belonging to the corresponding subgenotype was higher than 96%. Similarly, bootstrap values in phylogenetic trees supported clustering of reference strains with isolates belonging to the same subgenotypes. Moreover, both homology and phylogeny analyses showed that reference sequences had significant heterogeneity with isolates from other genotypes and subgenotypes. Sequence analysis further revealed that the mutation rate in the basal core promoter (BCP) region was extremely high in HBV/C2, relatively high in HBV/C1, but lower in HBV/C5 and HBV/C6. Mutations in the pre-core (Pre-C) region were common in HBV/C but the mutation rate was lower than in the BCP. HBV/ C5 has the oldest ancestral age, followed by C6, which is much more ancient than C1 and C2. This study successfully established references for HBV/C subgenotypes.

Hepatitis B virus mutations during mother-to-children transmission: a preliminary study

Objective To explore the mutations of hepatocellular carcinoma(HCC)-related hepatitis B virus(HBV)during mother-to-child transmission,so as to provide theoretic evidence for prophylaxis of HCC from the very beginning.Methods A total of 413 HBsAg-positive mothers and their newborns were enrolled in this study.Serum HBV DNA levels in maternal peripheral blood and cord blood of the newborns were measured using real-time quantitative PCR.Nested PCR together with cloning and sequencing methods were applied to examine the HCC-related HBV mutations in the preS and basal core promoter regions of HBV genome.All the newborns received standard HBV vaccination.Of the 413 newborns,104were successfully followed-up 7 months after birth,and the HBV mutations were examined if their circulating HBV DNA was detectable.Results Of the 413 newborns,41(9.9%)had HBV DNA level103 copies/mL in their cord blood.Four(3.8%)of the 104 newborns who were successfully followed up had circulating HBV DNA level103 copies/mL 7months after birth.Compared to mothers without HBV trans-placental transmission,those with HBV trans-placental transmission had no increase in HBV mutations in the basal core promoter region.However,the viral mutations containing T2898G/C,C3000 T,C3116T,T31 C,and T52 Cin the preS region of HBV subgenotype C2 significantly increased the risk of HBV trans-placental transmission(P0.05).The frequencies of the HCC-related mutations in the preS and basal core promoter regions of HBV genome were not significantly different between maternal peripheral blood and the cord blood of the newborns.Importantly,the HCC-related mutations were rarely found in the HBV-positive infants at 7months after birth.Conclusion The HBV mutations in the preS region of HBV subgenotype C2 may affect the trans-placental transmission of HBV.However,the quasispecies of HCC-related HBV mutants have no advantage in causing chronic HBV infection in infants.The HBV mutants which can promote HCC are selected during the long term chronic infection.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers.

In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

IDENTIFICATION OF X GENE MUTATIONS, GENOTYPE AND SUBGENOTYPE OF HEPATITIS B VIRUS (HBV)

Infection of hepatitis B virus (HBV) is a global health problem, including in Indonesia. There are currently an estimated 2 billion people worldwide are infected with HBV, about 75% of them are in Asia and 350 million of them will develop into chronic hepatitis B. Factors that influence the development of chronic hepatitis B into liver carcinoma include x gene mutation, HBV genotype and subgenotype. This research aims to identify x gene mutation, genotype and subgenotype of HBV infecting hepatitis B patients in Manado associated with the onset of liver carcinoma. HBV DNAs were isolated from blood samples of 30 hepatitis B patients. X gen was amplified using nested PCR with pre-designed primer pairs. Amplified DNA fragments were electrophoresed in 1.5% agarose and visualized under UV. DNA fragments were then separated and purified using Qiagen column, then sequenced to determine their nucleotide sequences of x gene. Amino acid of x protein were deduced from nucleotide sequence of x gene and used as basic to determine HBV genotype and subgenotype. X protein was aligned with those similar protein with the same subgenotype retrieved from GenBank to determine if there was a mutation at amino acid. The mutated x protein were compared with other mutation found in x protein in other literatures associated with the onset of liver carcinoma. Genotype and subgenotype of HBV isolated from blood samples of 10 patients was detected and showed that five patients were infected with B genotype HBV (2 patients were infected with B2 subgenotype, 2 patients with B3 subgenotype and 1 patient with B9 subgenotype). The rest of 5 patients were infected with C genotype HBV (1 patient with C1 subgenotype, 2 patients with C2 subgenotype, and 2 patients with C5 subgenotype). The mutation in x protein is related significantly to the clinical severity of the liver and hepatocellular carcinoma (HCC), ie V5L in subgenotype C2, and I127T and H94Y in subgenotype C5.

Distribution and diversity of hepatitis B virus genotypes in Yunnan, China.

Hepatitis B virus (HBV) is one of the most prevalent pathogens in the world, and infection with this virus is a serious threat for public health. Yunnan is considered as an important endemic center for blood-borne viruses such as human immunodeficiency virus and hepatitis C virus, in China. However, the distribution and diversity of HBV subgenotypes remain unclear in Yunnan province. In the current study, HBV positive samples were collected from different prefectures of Yunnan province and their molecular epidemiological characters were determined. Phylogenetic analysis on the pre-S/S gene (865 bps) showed the prevalence of four HBV genotypes, including genotype B (24 cases, 33.3%), genotype C (45 cases, 62.5%), genotype I (two cases, 2.78%) and C/D recombinants (one case, 1.39%). The most prevalent genotypes B and C could be sub classified into subgenotype B2 and C1, C2, C5, and C7, respectively. Clusters of subgenotype B2 and C2 consisted of strains from China and other East Asian countries, while subgenotype C1, C5, and C7 and genotype I formed a cluster together with strains from Southeast Asia. Using Bayesian inference from phylogenetic, HBV genotypes B and C were estimated to have originated in 1860s and 1910s with an evolutionary rate of 3.26 and 8.01 × 10−4 substitutions/site/year, respectively. These findings indicate that the distribution of HBV genotypes in Yunnan was influenced by strains from the rest of China and the neighboring countries. J. Med. Virol. 86: 1675–1682, 2014. © 2014 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants

In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

The Cross-Neutralizing Activity of Enterovirus 71 Subgenotype C4 Vaccines in Healthy Chinese Infants and Children

BackgroundEV71 is one of major etiologic causes of hand-foot-mouth disease (HFMD) and leads to severe neurological complications in young children and infants. Recently inactivated EV71 vaccines have been developed by five manufactures and clinically show good safety and immunogenicity. However, the cross-neutralizing activity of these vaccines remains unclear, and is of particular interest because RNA recombination is seen more frequently in EV71 epidemics.Methodology/Principal FindingsIn this post-hoc study, sera from a subset of 119 infants and children in two clinical trials of EV71 subgenotype C4 vaccines (ClinicalTrials.gov Identifier: NCT01313715 and NCT01273246), were detected for neutralizing antibody (NTAb) titres with sera from infected patients as controls. Cytopathogenic effect method was employed to test NTAb against EV71 subgenotype B4, B5, C2, C4 and C5, which were prominent epidemic strains worldwide over the past decade. To validate the accuracy of the results, ELISpot assay was employed in parallel to detect NTAb in all the post-vaccine sera. After two-dose vaccination, 49 out of 53 participants in initially seronegative group and 52 out of 53 participants in initially seropositive group showed less than 4-fold differences in NTAb titers against five EV71 strains, whereas corresponding values among sera from pediatric patients recovering from EV71-induced HFMD and subclinically infected participants were 8/8 and 41/43, respectively. The geometric mean titers of participants against five subgenotypes EV71 all grew significantly after vaccinations, irrespective of the baseline NTAb titer. The relative fold increase in antibody titers (NTAb-FI) against B4, B5, C2, and C5 displayed a positive correlation to the NTAb-FI against C4.Conclusions/SignificanceThe results demonstrated broad cross-neutralizing activity induced by two C4 EV71 vaccines in healthy Chinese infants and children. However, the degree of induced cross-protective immunity, and the potential escape evolution for EV71 still need to be monitored and researched in future for these new vaccines.

Genetic characterization of EV71 isolates from 2004 to 2010 reveals predominance and persistent circulation of the newly proposed genotype D and recent emergence of a distinct lineage of subgenotype C2 in Hong Kong

BackgroundEnterovirus 71 (EV71) is a common etiological agent of hand, foot and mouth disease (HFMD) in children. EV71 epidemics have been reported in Hong Kong in recent years, and yet the genetic information of EV71 strains circulating in our locality is limited. The objective of this study was to investigate the genetic evolution of these EV71 isolates in Hong Kong over a 7-year period.MethodsTwenty-two EV71 isolates from Hong Kong during 2004–2010 were included for phylogenetic analysis using partial VP2-VP3, 2C and 3D regions. Eight EV71 strains were selected for complete genome sequencing and recombination analysis.ResultsAmong the 22 EV71 isolates, 20 belonged to subgenotype C4 and 2 belonged to subgenotype C2 based on the phylogenetic analysis of partial VP2-VP3, 2C and 3D gene regions. Phylogenetic, similarity plot and bootscan analyses using complete genome sequences of seven EV71 isolates of subgenotype C4 supported that the “double-recombinant” strains of subgenotype C4 persistently circulating in Hong Kong should belong to a newly proposed genotype D. Further analysis revealed two clusters, subgenotypes C4b and C4a (proposed genotypes D1a and D1b respectively), with “genotype D1b” strains being predominant in recent years in Hong Kong. A distinct lineage of EV71 subgenotype C2 has emerged in Hong Kong in 2008. The evolutionary rate of EV71 was 3.1 × 10-3 nucleotide substitutions per site per year similar to that of other enterovirus, such as EV68, but was relatively lower than those of echovirus 30 and poliovirus. Molecular clock analysis using VP1 gene dated the time to the most recent common ancestor of all EV71 genotypes to 1900s, while the EV71 “double-recombinant” strains of “genotype D” were detected as early as 1998.ConclusionsThis study provides the molecular basis for proposing a new “genotype D” of EV71 and assigning a discrete lineage of subgenotype C2. EV71 strains of “genotype D” have been circulating in Hong Kong for over 7 years, with “genotype D1b” being predominant.

Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma, respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

High prevalence of hepatitis B virus genotype C/C1 in the Minangkabau ethnic group in Indonesia

BackgroundThe Minangkabau is one of the major ethnic groups in Indonesia. Previous studies with a limited number of samples have shown a different prevalence of HBV/C in the Minangkabau compared to the Indonesian population in general. The aim of this study was to assess the HBV genotype distribution pattern and the prevalence of pre-S, T1753V and A1762T/G1764A mutations among the Minangkabau HBV carriers. The samples were collected from Padang, West Sumatera and from western Java. Mixed primers for specific genotypes were used to determine the HBV genotype. Pre-S or S genes were amplified, sequenced and aligned with reference sequences from GenBank to derive a phylogenetic tree for subgenotyping. Pre-S genes were also analyzed for mutations. The basal core promoter (BCP) region was amplified and directly sequenced to analyze T1753V and A1762T/G1764A mutations.ResultsThe predominant HBV genotype among the Minangkabau HBV carriers (n=117) was C (72.6%) followed by B (24.8%) and co-infection with B and C (2.6%). The prevalence of pre-S mutations, including both the pre-S deletion and pre-S2 start codon mutation, was 41.0%, and the T1753V and A1762T/G1764A mutations were found in 51.9% and 71.2% respectively. HBV/C1 was the predominant HBV subgenotype in the Minangkabau HBV carriers, and was found in 66.2%, followed by B3, B7, C8, B2, B9, C2, and C10 (18.3%, 7.0%, 2.8%, 1.4%, 1.4%, 1.4%, and 1.4% respectively). From samples that were found to be co-infected with HBV B and C, two samples were successfully cloned and subgenotyped, including one with mixed subgenotypes of B3 and C1, and another one with mixed subgenotypes of B7, C1, putative intergenotypic of B/A, and C/A. Furthermore, three samples from donors of non-Minangkabau ethnicity from Padang were found to be infected with an intragenotypic recombination form, including a putative recombinant of B8/B3 and B9/B7.ConclusionHBV/C with subgenotype C1 was the predominant HBV genotype among HBV carriers of Minangkabau ethnicity. The prevalence of pre-S, A1762T/G1764A, and T1753V mutations was higher among the Minangkabau compared to Indonesian HBV carriers in general.

Accumulation of the Mutations in Basal Core Promoter of Hepatitis B Virus Subgenotype C1 Increase the Risk of Hepatocellular Carcinoma in Southern China

Hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, HBV subgenotype C1 is the major subgenotype in Southern China. The aim of this study was to investigate whether there was the specific mutation patterns in HBV/C1 associated with Southern Chinese patients with HCC. Mutations in HBV basal core promoter (BCP) and their association with HCC were assessed in a matched cross-sectional control study of 102 HCC and 105 chronic hepatitis (CH) patients (from Guangdong, China) infected with HBV/C1. Functional analysis of HBx mutants was performed by the colony formation assay and the luciferase assays. T1762/A1764 double mutations was frequently found in patients infected with HBV/C1, regardless of clinical status (64.7% in HCC and 51.4% in CH, P>0.05). Unexpectedly, the adjacent V1753 or A1768 mutation significantly increased the risk of HCC (P<0.05). Moreover, the prevalence of triple or quadruple mutations in BCP was significantly higher in patients with HCC than those with CH, particularly for HBeAg-positive-carriers (P<0.05). Functional analysis revealed that T1762/A1764 mutation alone did not alter the transcriptional activity and the inhibitory effects on cell proliferation of HBx, but triple or quadruple mutations largely abrogated this effect. Accumulation of mutations involving V1753 or/and A1768 in addition to T1762/A1764 in BCP region were closely related to HCC among the patients infected with HBV/C1, particularly for HBeAg-positive-carriers. The increased risk of HCC caused by BCP variants may be attributable partially to modifying the biological functions of HBx.