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Genotype I of hepatitis B virus (HBV) was proposed recently following sequencing of complete HBV genomes from Vietnam and Laos. However, its long-term molecular evolution is unknown. The objectives of this study were to study the molecular evolution of this genotype from an asymptomatic HBsAg carrier from the Long An cohort over a 15-year period was studied using both NGS and clone-based sequencing. The number of complete genome sequences obtained in 2004, 2007, 2013, and 2019 are 17, 20, 19, and 10, respectively. All strains belong to subgenotype I1, except for six (five from 2007 and one from 2019) and 8 further strains from 2007 which form a cluster branching out from other subgenotype I sequences, supported by a 100% bootstrap value. Based on complete genome sequences, all of the estimated intragroup nucleotide divergence values between these strains and HBV subgenotypes I1-I2 exceed 4%. These strains are recombinants between genotype I1 and subgenotype C but the breakpoints vary. The median intrahost viral evolutionary rate in this carrier was 3.88E-4 substitutions per site per year. The Shannon entropy (Sn) ranged from 0.55 to 0.88 and the genetic diversity, D, ranged from 0.0022 to 0.0041. In conclusion, our data provide evidence of novel subgenotypes. Considering that the 8 strains disappeared after 2007, while one of the 6 strains appears again in 2019, we propose these 6 strains as a new subgenotype, provisionally designated HBV subgenotype I3 and the 8 strains as aberrant genotype., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A → T) and 1764(G → A) and a stop mutation at nt1896(G → A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Serum osteopontin (OPN) concentrations were found to be significantly increased in patients infected with hepatitis B virus (HBV) and patients with hepatocellular carcinoma (HCC)., Objective: The aim of this study was to determine the association among HCC, OPN, and HBV., Methods: Two hundred and forty-one subjects were recruited and divided into 6 groups: healthy controls, asymptomatic HBsAg carriers, HBsAg (-) patients with other tumors, HBsAg (+) chronic liver disease patients, HBsAg (+) patients with HCC, and HBsAg (-) patients with HCC or liver cirrhosis (LC). Serum concentrations of OPN and HBsAg were measured and analyzed., Results: OPN concentrations in the HBsAg (+) HCC group were significantly higher than the healthy control group and the HBsAg (-) patients with other cancers (both p = 0.0001). The OPN concentrations of the HBsAg (-) patients with HCC or LC also did not differ significantly from those of the healthy control group (p = 0.075). There is a correlation between the titer of HBsAg and concentrations of OPN in all 3 HBsAg (+) groups (all p values <0.05)., Conclusions: Infection with HBV may increase the serum concentrations of OPN. The association of OPN and HCC may be not attributable to tumor development per se but, rather, to HBV infection., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1-D10 exceed 4 %. Compared to the other subgenotypes (D1-D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.

Objectives: We aimed to determine the prevalence of hepatitis B virus (HBV) drug-resistant mutations in patients co- infected with HBV/human immunodeficiency virus (HIV), including both drug-naïve subjects and those who received antiretroviral therapy (ART) in Guangxi, where the prevalence of HIV/HBV co-infection is highest in China., Methods: Two hundred and three subjects co-infected with HBV/HIV were recruited, including 123 drug-naïve patients (group 1) and 80 who received ART (group 2). The polymerase gene of HBV in the serum of all study subjects was analysed., Results: The results showed that the prevalence of HBV drug-resistant mutations in group 2 (76.5%, 95% CI 56.3-96.7) was significantly higher than that in group 1 (1.4%, 95% CI -1.4 to 4.2; χ2 = 50.955, p < 0.05). The major pattern of lamivudine (3TC)-resistant mutations is L180M+M204I+L80I (35.7%). In total, 95% of subjects with resistant mutations had cross-resistance to telbivudine and entecavir. No putative tenofovir disoproxil fumarate (TDF) resistance change was found. Five subjects (6.5%) in group 2 had HBV viral loads over 10 × 106 copies/mL. Four of them had 3TC-resistant mutations. Multivariate analysis showed that ART was the only factor associated with the development of drug-resistant mutations., Conclusion: Treating HIV in HIV/HBV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistant mutations. TDF could not completely suppress HBV replication., (© 2018 S. Karger AG, Basel.)

In the era of combination therapy for human immunodeficiency virus (HIV), liver disease including hepatocellular carcinoma (HCC), are the major causes of death for patients co-infected with hepatitis B virus (HBV) and HIV. However, the mechanisms remain obscure. We aimed to determine whether HCC-related HBV mutations including 1762T/1764A double mutation and pre-S deletions occur more frequently in HBV/HIV co-infected individuals compared to HBV mono-infected individuals. In this study, the basic core promoter (BCP) and the preS/S regions of HBV isolated from 61 pairs of HBV/HIV co-infected and HBV mono-infected participants were analyzed. We found that the prevalence of HBV isolates with 1762T/1764A and/or preS deletion mutations was 37.7% (95% CI: 29.1-46.3). The prevalence of these mutations in HBV/HIV co-infected group (52.5%, 95% CI: 40.0-65.0) was significantly higher than in the HBV mono-infected group (23.0%, 95% CI: 12.4-33.6) (X 2 =11.307, P<0.05). HBV/HIV co-infection was associated with higher viral loads but these higher viral loads were not associated with the higher prevalence of HCC-related HBV mutations. Individually 1762T1764A (44.3%) or preS deletions (23%) occurred more frequently in isolates from co-infected compared to mono-infected individuals (21.3%, 4.9%, respectively) (X 2 =7.290, P<0.05; X 2 =8.270, P<0.05). Moreover, 1762T/1764A and preS deletions occurred more frequently in genotypes C and I compared to genotype B (p<0.05). Multivariate analysis revealed that co-infection with HIV was associated with the development of both 1762T/1764A ((RR: 2.932(1.325-6.488)) and preS deletions ((RR: 5.759(1.562-21.235)). These results demonstrate that co-infection with HIV was associated with increased prevalence of HCC-related mutations in HBV isolates from Chinese patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Background: Although persistent hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), the mechanisms of oncogenesis remain obscure., Aims: To determine whether the findings that HBV basal core promoter (BCP) A1762T, G1764A double mutations, pre-S deletions and a combination of both are risk factors of HCC are supported by geographical epidemiology., Methods: Study subjects were recruited from Long An county, where the incidence of HCC is the highest, and five other counties in Guangxi, where the HCC incidence is lower and varies among them. The Pre-S region and BCP of HBV from all study subjects were amplified and sequenced and the data were analysed using chi-squared tests., Results: The prevalence of BCP and pre-S mutations differs significantly (χ(2) = 9.850, 5.135, respectively, all P < 0.01) between Long An and the other counties. However, the prevalence of combined BCP and pre-S mutations does not differ significantly (χ(2) = 1.510, P > 0.05). These mutations are less frequent in the young but the prevalence of pre-S deletions does not increase with age. The prevalence of these mutations does not differ significantly between men and women but is significantly higher in Zhuang than the other ethnic populations. Among the other five counties, the prevalence of BCP mutations in counties where the HCC incidence is high is significantly higher than that of counties where the HCC incidence is low., Conclusions: Combined BCP double mutations and pre-S deletion may not increase the risk of HCC, although these mutations are a risk factor of HCC when they present alone., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sequencing of the complete hepatitis B virus (HBV) genomes from Vietnam, China and Laos led to the identification of a complex recombinant, referred to initially as an aberrant genotype and later proposed to be a new genotype, I. However, epidemiological data regarding this new genotype are lacking. A cross-sectional study was carried out to investigate the epidemiology of HBV candidate genotype I in Guangxi, China using stratified, random cluster sampling. Four thousand five hundred thirteen subjects were recruited from five counties within Guangxi. Three genotypes, B, C, and I, were identified with a prevalence of 32.6% (114/350), 64% (224/350), and 3.4% (12/350), respectively. All the genotype I isolates belong to candidate subgenotype I1 and were found in Bing Yang (15.3%, 9/59) and Na Po (5.0%, 3/60) counties only. The prevalence of this subgenotype is significantly higher in males (5.1%, 10/195) than in females (1.3%, 2/155; X(2)  = 3.959, P < 0.05) but does not differ significantly with age. It was found in the Han (4.5%, 9/201) and Zhuang (3.1%, 3/97) ethnic populations only. There is no significant difference from other genotypes in the prevalence of HBV serological markers. Phylogeographic analysis revealed that genotype I1 likely arose in Long An county, then spread later to Bing Yang, Na Po counties and elsewhere in southeast Asia. In conclusion, the distribution of candidate genotype I within Guangxi is not even and it is highly endemic in some counties. Its prevalence is associated with gender and ethnicity. Subgenotype I1 likely originated in Long An county., (Copyright © 2013 Wiley Periodicals, Inc.)

The Chinese national goals for control of hepatitis B virus (HBV) infection were to achieve a prevalence of HBsAg below 7% for the entire population, and 1% for children under 5-year old, by 2010. To determine whether Guangxi, a multi-minority province with a low socio-economic status and a very high prevalence of HBV, achieved this goal, a seroepidemiological survey of HBV infection was carried out using stratified, random cluster sampling. The results show that the overall prevalence of HBsAg is 9.16% [95% confidence interval (CI) = 8.32-10%]. The prevalence in males (10.96%, 95% CI = 9.64-12.28%) is significantly higher than in females (7.71%, 95% CI = 6.64-8.78%; χ(2) = 10.5923, P < 0.05). The prevalence in children under 5-year old is 3.62% (95% CI = 0.60-6.64%) and increases with age. The prevalence of HBsAg in non-immunized individuals is significantly higher than in those immunized completely, although not within 24 hr of birth (χ(2) = 31.426, P < 0.05); a significant difference was found in those below the age of 20 years but not in older persons. Gender, age, immunization history, and familial HBsAg carriers are risk factors for infection. In conclusion, this study indicates that Guangxi has not reached the goal for the control of HBV infection. Catch-up HBV immunization may not protect adults effectively against infection in highly endemic regions., (Copyright © 2012 Wiley Periodicals, Inc.)

Recently, a complex (X/C) hepatitis B virus (HBV) recombinant, first reported in 2000, was proposed as a new genotype; although this was refuted immediately because the strains differ by less than 8 % in nucleotide distance from genotype C. Over 13.5 % (38/281) of HBV isolates from the Long An cohort in China were not assigned to a specific genotype, using current genotyping tools to analyse surface ORF sequences, and these have about 98 % similarity to the X/C recombinants. To determine whether this close identity extends to the full-length sequences and to investigate the evolutionary history of the Long An X/C recombinants, 17 complete genome sequences were determined. They are highly similar (96-99 %) to the Vietnamese strains and, although some reach or exceed 8 % nucleotide sequence difference from all known genotypes, they cluster together in the same clade, separating in a phylogenetic tree from the genotype C branch. Analysis of recombination reveals that all but one of the Long An isolates resembles the Vietnamese isolates in that they result from apparent recombination between genotype C and a parent of unknown genotype (X), which shows similarity in part to genotype G. The exception, isolate QL523, has a greater proportion of genotype C parent. Phylogeographic analysis reveals that these recombinants probably arose in southern China and spread later to Vietnam and Laos.

Objective: To determine the relationship between the serum hepatitis B virus (HBV) DNA and the risk of primary liver cancer (PLC)., Methods: Farmers aged 30 to 55 years in Long An county were recruited in this study Blood samples were collected and the sera were tested for HBsAg using Enzyme-Linked ImmunoSorbent Assay (ELISA), and the HBsAg-positive sera were further tested for viral DNA using nested polymerase chain reaction (nPCR). The study subjects were divided into three groups. The first group was positive for both HBsAg and HBV DNA. The second group was positive for HBsAg but negative for HBV DNA. Age-, sex-, residence-matched HBsAg negative controls for group 1 and group 2 were enrolled in the third group. The cohort was followed up for four years., Results: The positive rate of HBsAg in these farmers was 14.52% (3975/27,379), and the HBV DNA positive rate in HBsAg positive subjects was 40.35% (1604/3975). The total PLC incidence rate in Group 1 and 2 was 672.45 /100,000 person-years (PY), significantly higher than that in Group3 (17.19 /100,000 PY). The relative risk (RR) was 39.123, and the 95% confidence interval (CI) was 9.018-159.146. The PLC incidence rate of Group 1 (984.03/100,000 PY) was significantly higher than that of Group2 (324.38 /100,000 PY). The RR was 3.034, and the 95% CI was 1.795-5.125. Multivariate analyses of Group1 and 2 with Cox model showed that sex, age, serum HBV DNA, and family history of PLC were independent risk factors of PLC., Conclusion: HBV DNA and HBsAg positive subjects have a higher chance to develop PLC than HBV DNA negative-, HBsAg positive subjects.

Background/aims: Although there have been a few reports regarding the effect of basal core promoter (BCP) double mutations (A1762T and G1764A) on hepatitis B viral loads, the association remains uncertain. We aim to determine the association after controlling for HBeAg - a strong confounding factor., Methods: We selected randomly 190 individuals from a Chinese cohort of 2258 subjects for cross-sectional analysis and 56 of the 190 for longitudinal analysis of viral loads., Results: In multivariable analysis of the cross-sectional data, BCP double mutations are significantly associated with lower viral loads in HBeAg positive subjects but no difference was found in anti-HBe positive subjects. Triple mutations at nucleotide (nt) 1753, 1762 and 1764 and mutations between nt 1809 and 1817, precore stop mutation (nt 1896) and genotype are not associated with viral loads in either HBeAg or anti-HBe positive subjects. Analysis of the longitudinal data yielded similar results to the cross-sectional data. Viral loads differ significantly between individuals infected with wild-type and BCP double mutations prior to HBeAg seroconversion but this difference is lost after seroconversion., Conclusions: BCP double mutations are associated with lower viral loads in HBeAg positive individuals but have no effect on the viral loads of anti-HBe positive individuals.

A matched nested case-control study of 33 paired cases and controls was conducted, based on a study cohort in Long An county, Guangxi, China, to determine whether infection with hepatitis B virus (HBV) with pre-S deletions is independently associated with the development of hepatocellular carcinoma (HCC), without the confounding effects of basal core promoter (BCP) double mutations. The prevalence of pre-S deletions was significantly higher in HCC (45.5 %, 15 of 33) than the controls (18.2 %, 6 of 33) (P<0.01), under the control of the influence of BCP double mutations. Most of the pre-S deletions occurred in, or involved, the 5' half of the pre-S2 region and the difference between HCC (93.3 %, 14 of 15) and controls (66.7 %, four of six) was significant for this region (P=0.015). There was no significant difference in pre-S deletions between the BCP mutant group and BCP wild-type group (P>0.05), nor was the prevalence of pre-S deletions significantly different between genotypes B and C (P>0.1). These results suggest that pre-S deletions constitute an independent risk factor for HCC and their emergence and effect are independent of BCP mutations. The 5' terminus of pre-S2 is the favoured site for the deletion mutations, especially in HCC cases. Further prospective studies are required to confirm the role of these mutations in the development of HCC.