Characterization of BCP/PreC/C region quasispecies in treatment-naive patients with different phases of HBV infection using next-generation sequencing.

BACKGROUND: To analysis of quasispecies (QS) changes and high-frequency mutations in the BCP/PreC/C region of patients at different phases of hepatitis B virus (HBV) infection and provides novel biomarkers for the diagnosis of chronic hepatitis B (CHB) patients. METHODS: With the application of next-generation sequencing technology, we were able to sequence the HBV BCP/PreC/C regions in 40 patients, each at different phases of the HBV infection. The heterogeneity of QS and the frequency of mutations were calculated using MEGA 7 software. RESULTS: Our results show that the complexity and diversity of the BCP/PreC/C QS in HBeAg-positive CHB patients are significantly higher than those in HBeAg-positive chronic infection patients, while HBeAg-negative chronic infection patients had significantly higher QS complexity and diversity than HBeAg-negative CHB patients. In addition, HBeAg-negative patients showed reduced complexity but increased diversity compared with HBeAg-positive patients. Receiver operating characteristic curves showed that G1764A, C2102T, dN and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-positive CHB, while the A2189C, dS and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-negative chronic hepatitis. Finally, our study also found that G1896A and A2159G may be hotspot mutations affecting HBeAg seroconversion. CONCLUSION: Our research elucidates the evolution of HBV by analyzing QS heterogeneity and mutation patterns, offering novel serum biomarkers for enhancing clinical diagnosis and disease prognosis. This comprehensive approach sheds light on the intricate dynamics of HBV progression and paves the way for more precise medical interventions.

The distinct spatiotemporal evolutionary landscape of HBV and HDV largely determines the unique epidemic features of HDV globally.

Chronic infection of hepatitis B virus (HBV) and hepatitis D virus (HDV) causes the most severe form of viral hepatitis. Due to the dependence on HBV, HDV was deemed to co-evolve and co-migrate with HBV. However, we previously found that the naturally occurred HDV/HBV combinations do not always reflect the most efficient virological adaptation (Wang et al., 2021). Moreover, regions with heavy HBV burden do not always correlate with high HDV prevalence (e.g., East Asia), and vice versa (e.g., Central Asia). Herein, we systematically elucidated the spatiotemporal evolutionary landscape of HDV to understand the unique epidemic features of HDV. We found that the MRCA of HDV was from South America around the late 13th century, was globally dispersed mainly via Central Asia, and evolved into eight genotypes from the 19th to 20th century. In contrast, the MRCA of HBV was from Europe ∼23.7 thousand years ago (Kya), globally dispersed mainly via Africa and East Asia, and evolved into eight genotypes ∼1100 years ago. When HDV stepped in, all present-day HBV genotypes had already formed and its global genotypic distribution had stayed stable geographically. Nevertheless, regionalized HDV adapted to local HBV genotypes and human lineages, contributing to the global geographical separation of HDV genotypes. Additionally, a sharp increase in HDV infections was observed after the 20th century. In conclusion, HDV exhibited a distinct spatiotemporal distribution path compared with HBV. This unique evolutionary relationship largely fostered the unique epidemic features we observe nowadays. Moreover, HDV infections may continue to ramp up globally, thus more efforts are urgently needed to combat this disease.

Differences between Chronically Hepatitis B Virus-Infected Pregnant Women with and without Intrafamilial Infection: From Viral Gene Sequences to Clinical Manifestations.

INTRODUCTION: This study aimed to investigate the differences between pregnant women with chronic hepatitis B virus (HBV) infection and intrafamilial infection and those without intrafamilial infection. METHODS: HBV-DNA was extracted from the sera of 16 pregnant women with chronic hepatitis B (CHB) and their family members for gene sequencing and phylogenetic analyses. A total of 74 pregnant women with CHB were followed up from the second trimester to 3 months postpartum. Viral markers and other laboratory indicators were compared between pregnant women with CHB with and without intrafamilial infection. RESULTS: The phylogenetic tree showed that HBV lines in the mother-spread pedigree shared a node, whereas there was an unrelated genetic background for HBV lines in individuals without intrafamilial infection. From delivery to 3 months postpartum, compared with those without intrafamilial infection, pregnant women with intrafamilial infection were related negatively to HBV-DNA (ß = -0.43, 95% confidence interval [CI]: -0.76 to -0.12, p = 0.009), HBeAg (ß = -195.15, 95% CI: -366.35 to -23.96, p = 0.027), and hemoglobin changes (ß = -8.09, 95% CI: -15.54 to -0.64, p = 0.035) and positively to changes in the levels of alanine aminotransferase (ß = 73.9, 95% CI: 38.92-108.95, p < 0.001) and albumin (ß = 2.73, 95% CI: 0.23-5.23, p = 0.033). CONCLUSION: The mother-spread pedigree spread model differs from that of non-intrafamilial infections. Pregnant women with intrafamilial HBV infection have less hepatitis flares and liver damage, but their HBV-DNA and HBeAg levels rebound faster after delivery, than those without intrafamilial infection by the virus.

Ancient hepatitis B viruses from the Bronze Age to the Medieval period.

Hepatitis B virus (HBV) is a major cause of human hepatitis. There is considerable uncertainty about the timescale of its evolution and its association with humans. Here we present 12 full or partial ancient HBV genomes that are between approximately 0.8 and 4.5 thousand years old. The ancient sequences group either within or in a sister relationship with extant human or other ape HBV clades. Generally, the genome properties follow those of modern HBV. The root of the HBV tree is projected to between 8.6 and 20.9 thousand years ago, and we estimate a substitution rate of 8.04 × 10-6-1.51 × 10-5 nucleotide substitutions per site per year. In several cases, the geographical locations of the ancient genotypes do not match present-day distributions. Genotypes that today are typical of Africa and Asia, and a subgenotype from India, are shown to have an early Eurasian presence. The geographical and temporal patterns that we observe in ancient and modern HBV genotypes are compatible with well-documented human migrations during the Bronze and Iron Ages1,2. We provide evidence for the creation of HBV genotype A via recombination, and for a long-term association of modern HBV genotypes with humans, including the discovery of a human genotype that is now extinct. These data expose a complexity of HBV evolution that is not evident when considering modern sequences alone.

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription.

Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

Evolutionary history of hepatitis B virus genotype H.

Hepatitis B virus genotype H (HBV-H) molecular evolution was studied by comparing all published whole-genome sequences. Bayesian coalescent analysis was performed to estimate phylogenetic relationships, time to the most recent common ancestor (tMRCA), and viral population dynamics along the time. Phylogenetic tree demonstrated two main clades or lineages: HBV-H I (with sequences from Central and North America) and HBV-H II (with sequences from North and South America, and Asia). HBV-H II had more genome sequences (n = 26; 83.9%), including one specific subclade with all sequences outside of the Americas. Overall HBV-H tMRCA dated back to 1933 (95% highest posterior density interval [HPD 95%]: 1875-1957) with a very probable origin in Mexico and posterior dissemination to other American and Asian countries. The temporal analysis demonstrated that HBV-H I spread only in Mexico and the neighbor country of Nicaragua probably in the 1960s to the 1970s (1968; HPD 95%: 1908-1981), while HBV-II disseminated to other American and Asian countries around one decade later (1977; HPD 95%: 1925-1985). The phylogeographic analysis reinforced the Mexican origin of this genotype. The whole HBV-H population increased from the 1980s to the 2000s. In conclusion, HBV-H has two main lineages with a common origin in Mexico approximately nine decades ago.

A3G-induced mutations show a low prevalence and exhibit plus-strand regional distribution in hepatitis B virus DNA from patients with non-hepatocellular carcinoma (HCC) and HCC.

APOBEC3G (A3G) cytidine deaminase is an innate immune restriction factor that can edit and inhibit hepatitis B virus (HBV) replication. The preferred target of A3G is deamination of the third cytosine of 5'CCC to form a mutant marker 5'CC C → K. However, the distribution of A3G-induced mutations on HBV DNA during infection is not well characterized. To provide clarity, we obtained the HBV DNA sequences from HBV infected individuals with and without hepatocellular carcinoma (HCC and non-HCC, respectively), from the NCBI database, and calculated the r values of A3G-induced 5'CC C → K mutation prevalence in HBV DNA. A3G-induced mutations were weakly prevalent and mainly distributed in the plus strand of HBV DNA (r = 1.407). The mutations on the minus strand were weaker (r = .8189). There were A3G-induced mutation regions in the 1200 to 2000 nt region of the plus strand and the 1600 to 1500 nt region of the minus strand. There was no significant difference in the r values of A3G-induced mutations in HBV DNA between the HCC and non-HCC groups. However, the rvalue of the plus strand 2400 to 2800 nt regions of HCC derived HBV DNA (r = 4.2) was significantly higher than that of the same regions of non-HCC derived HBV DNA (r = 1.21). These findings clarify the weak prevalence and preferred plus-strand distribution of A3G-induced mutations on HBV DNA from HCC and non-HCC. These findings may provide valuable clues regarding the interaction mechanism between A3G and HBV DNA and inform HCC screening.

Rapid, random-access, and quantification of hepatitis B virus using the Cepheid Xpert HBV viral load assay.

BACKGROUND: Monitoring viral load (VL) is an essential part of the management of patients chronically infected with hepatitis B virus (HBV). The commercial HBV VL assays currently available are generally performed on high-throughput platforms for batch wise testing of plasma samples, with relatively long turn-around-times. Rapid VL testing could provide immediate input to clinical decision making. METHODS: One hundred two stored plasma samples from 102 patients who were previously tested for HBV VL by the Cobas Ampliprep/Taqman or Cobas 4800 (Roche, Pleasanton, CA), were analyzed by the recently introduced Cepheid Xpert HBV Viral Load Assay. Thirty-one of the 102 samples were negative for HBV DNA and 71 out of 102 samples had a detectable VL. HBV DNA loads ranged from <20 to 5E8 IU/mL. HBV genotypes (A, B, C, D, E, and G) were known for 52 of the VL positive samples. Correlation of VL results between both assays was determined by the Pearson correlation coefficient (r2 ). The level of concordance was assessed using the Bland-Altman analysis. RESULTS: HBV VLs correlated well between both assays, across all genotypes (Pearson correlation coefficient r2 = 0.987). Six samples exceeded a 0.5 log difference between assays. Bland-Altman analysis demonstrated a mean of the difference of -0.107 log and a standard deviation of 0.271 log. CONCLUSION: High correlation was observed between the Roche Cobas HBV Viral Load tests and the Xpert HBV Viral Load Assay, thus enabling rapid, random access, and accurate HBV VL assessment.

Applicability of oral fluid samples for tracking hepatitis B virus mutations, genotyping, and phylogenetic analysis.

Little is known about the usefulness of saliva samples for hepatitis B virus (HBV) genotyping and mutation analysis. The aim of this study was to evaluate the usefulness of oral fluid samples to determine HBV genotype distribution, S/polymerase mutations, and HBV subpopulation diversity among chronically HBV-infected individuals. Serum and oral fluid samples were obtained from 18 individuals for PCR and nucleotide sequencing of the HBV surface antigen gene. Biochemical analysis of liver enzymes (ALT, AST, GGT) and HBV, HCV, and HIV serological tests were also performed. All serum samples were HBsAg (+), anti-HBc (+), and anti-HBs (-); 55.6% were HBeAg (+)/anti-HBe (-), and 11.1% were anti-HIV (+). The mean HBV DNA viral load was 6.1 ± 2.3 log IU/mL. The HBV genotype distribution was as follows: A, 72.2%; D, 11.1%; E, 5.6%; F, 11.1%. A concordance of 100% in genotype classification and 99.8% in sequence similarity between paired oral fluid and serum samples was observed. HBsAg mutations were detected in all samples, but no resistance mutations were found in the polymerase gene. This study demonstrates that oral fluid samples can be used reliably for tracking HBV mutations, genotyping, and phylogenetic analysis. This could be important for molecular epidemiology studies with hard-to-reach populations.

Molecular epidemiology of hepatitis B virus genotypes and subgenotypes in ethnic minority populations, Yunnan province, China.

The aim of our study was to determine the distribution of hepatitis B virus (HBV) genotypes and subgenotypes in ethnic minorities in Yunnan province to provide evidence supporting the theoretical basis for hepatitis B prevention and control. We obtained serum samples and demographic data from 765 individuals reported by Yunnan province who had either acute or chronic HBV infection and were from one of 20 ethnic minority populations: Achang, Bai, Brown, Tibetan, Dai, Deang, Dulong, Hani, Hui, Jingpo, Lahu, Yi, Lisu Miao, Naxi, Nu, Pumi, Wa, Yao, or Zhuang people. We sequenced the HBV DNA and determined the genotypes and subgenotypes of the isolated HBVs. We mapped the genotype and subgenotype distribution by ethnic minority population and conducted descriptive analyses. There were four genotypes among the 20 ethnic groups: genotype B (21.3% of samples), C (76.6%), D (1.8%) and I (0.3%). The most common subgenotype was C1. There were no genotype differences by gender (P = 0.954) or age (P = 0.274), but there were differences by region (P < 0.001). There were differences in genotype distribution (P < 0.001) and subgenotype distribution (P = 0.011) by ethnic group. Genotype D was most prominent in Tibet and most HBV isolates were C/D recombinant viruses. The only two genotype I virus isolates were in Zhuang people. Susceptibility and geographic patterns may influence HBV prevalence in different ethnic populations, but additional research is needed for such a determination.

Deep sequencing of HBV pre-S region reveals high heterogeneity of HBV genotypes and associations of word pattern frequencies with HCC.

Hepatitis B virus (HBV) infection is a common problem in the world, especially in China. More than 60-80% of hepatocellular carcinoma (HCC) cases can be attributed to HBV infection in high HBV prevalent regions. Although traditional Sanger sequencing has been extensively used to investigate HBV sequences, NGS is becoming more commonly used. Further, it is unknown whether word pattern frequencies of HBV reads by Next Generation Sequencing (NGS) can be used to investigate HBV genotypes and predict HCC status. In this study, we used NGS to sequence the pre-S region of the HBV sequence of 94 HCC patients and 45 chronic HBV (CHB) infected individuals. Word pattern frequencies among the sequence data of all individuals were calculated and compared using the Manhattan distance. The individuals were grouped using principal coordinate analysis (PCoA) and hierarchical clustering. Word pattern frequencies were also used to build prediction models for HCC status using both K-nearest neighbors (KNN) and support vector machine (SVM). We showed the extremely high power of analyzing HBV sequences using word patterns. Our key findings include that the first principal coordinate of the PCoA analysis was highly associated with the fraction of genotype B (or C) sequences and the second principal coordinate was significantly associated with the probability of having HCC. Hierarchical clustering first groups the individuals according to their major genotypes followed by their HCC status. Using cross-validation, high area under the receiver operational characteristic curve (AUC) of around 0.88 for KNN and 0.92 for SVM were obtained. In the independent data set of 46 HCC patients and 31 CHB individuals, a good AUC score of 0.77 was obtained using SVM. It was further shown that 3000 reads for each individual can yield stable prediction results for SVM. Thus, another key finding is that word patterns can be used to predict HCC status with high accuracy. Therefore, our study shows clearly that word pattern frequencies of HBV sequences contain much information about the composition of different HBV genotypes and the HCC status of an individual.

Genetic diversity of hepatitis B virus in Yunnan, China: identification of novel subgenotype C17, an intergenotypic B/I recombinant, and B/C recombinants.

Yunnan is considered to be a geographical hotspot for the introduction, mutation and recombination of several viruses in China. However, there are limited data regarding the genotypic profiles of hepatitis B virus (HBV) in this region. In this study, we characterized 206 HBV strains isolated from chronic hepatitis B patients in Yunnan, China. Initial genotyping based on 1.5 kb sequences revealed that genotype C was the most prevalent at 52.4 % (108/206), followed by genotype B at 30.6 % (63/206) and unclassified genotypes at 17.0 % (35/206). To characterize the 35 unclassified strains, 32 complete HBV genomes were amplified and analysed; 17 isolates were classified within a known subgenotype, 8 were classified as B/C recombinants, 1 was classified as a B/I recombinant and 6 constituted a potentially novel C subgenotype that we designated as C17, based on the characteristics of a monophyletic cluster, >4 % genetic distances, no significant evidence of recombination and no epidemiological link among individuals. Thus, multiple subgenotypes - namely B1, B2, B4, C1, C2, C3, C4, C8 and C17 - and two distinct intergenotypic recombinants exist in Yunnan, China, highlighting the complex and diverse distribution pattern of HBV genotypic profiles.

Distribution and difference of APOBEC-induced mutations in the TpCpW context of HBV DNA between HCC and non-HCC.

Hepatitis B virus (HBV) DNA is vulnerable to editing by human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. However, the distribution of APOBEC-induced mutations on HBV DNA is not well characterized. To this end, we obtained the HBV DNA sequence of HBV-infected individuals with and without hepatocellular carcinoma (HCC and non-HCC groups, respectively) from NCBI database and calculated the rapo values of APOBEC-induced TpCpW→TpKpW mutation prevalence in HBV DNA. The results showed that the APOBEC-induced mutations were mainly distributed in the minus strand of non-HCC-derived HBV DNA (rapo = 2.04), while the mutation on the plus-strand was weaker (rapo = 0.99). There were high APOBEC-induced mutation regions in the minus strand of HBV DNA 1 to 1000 nucleotides (nts) region and in the plus-strand of HBV DNA 1000 to 1500 nts region; the mutations in the 1 to 1000 nts region were mainly TpCpW→TpTpW mutation types (total T/G: 111/18) and a number of these were missense mutations (missense/synonymous: 35/94 in P gene, 17/15 in S gene, and 5/10 in X gene). The difference between minus to plus-strand rapo of HCC-derived HBV DNA (1.96) was greater than that of the non-HCC group (1.05). The minus-strand rapo of HCC-derived HBV DNA regions 1000 to1500nts and 1500 to 2000 nts (rapo = 4.2 and 4.2) was also higher than that of the same regions of non-HCC-derived HBV DNA (rapo = 1.2 and 1.1). Finally, the ratio of minus to plus-strand rapo was used to distinguish HCC-derived HBV DNA from non-HCC-derived HBV DNA. This study unraveled the distribution characteristics of APOBEC-induced mutations on double strands of HBV DNA from HCC and non-HCC samples. Our findings would help understand the mechanism of APOBECs on HBV DNA and may provide important insights for the screening of HCC.

An Association Between Core Mutations in Hepatitis B Virus Genotype F1b and Hepatocellular Carcinoma in Alaskan Native People.

Hepatitis B virus (HBV) genotype F1b infection is strongly associated with hepatocellular carcinoma (HCC) in young Alaskan Native (AN) people. However, the mechanisms by which genotype F1b causes HCC are unclear. Here, we analyzed the clinical and virological significance of genotype F1b in long-term serial samples from 20 HCC patients with HBV infection. Complete sequence analyses revealed that all isolates were genotype F1b. In the HCC patients, T1938C and A2051C mutations in the core region had accumulated significantly with A1762T/G1764A mutations in the basal core promoter (BCP) region and G1896A mutation in the precore (PC) region. Several HBV clones containing the core mutations were examined for their replication efficiency and core stability in vitro. Clones containing the A2051C mutation replicated more efficiently than the wild type in association with enhanced stability of core protein dimerization. In chimeric mice with human hepatocytes carrying BCP/PC/2051 mutant but not with wild-type virus, liver fibrosis was induced in association with high levels of serum HBV DNA and hepatitis B surface antigen. Interestingly, microarray analysis and validation study showed that five genes associated with cell proliferation or carcinogenesis, v-myc avian myelocytomatosis viral oncogene homolog, Grb2-associated binding protein 2, bradykinin receptor B2, follistatin, and mitogen-activated protein kinase kinase kinase 8, were significantly up-regulated in human hepatocytes infected with genotype F1b, particularly the BCP/PC/2051 mutant, compared with other genotypes. Conclusion: We have identified an association between Alaska-specific core mutations and HCC development in AN people infected with genotype F1b; accumulation of these core mutations during the course of chronic infection with genotype F1b would contribute to HCC development in AN people earlier in life.

Seroprevalence and genotypic characterization of HBV among low risk voluntary blood donors in Nairobi, Kenya.

BACKGROUND: Hepatitis B virus (HBV) causes significant morbidity and mortality globally primarily due to its ability to cause hepatitis, liver cirrhosis and hepatocellular carcinoma. The Kenya National Blood Transfusion Services screens for Hepatitis B antibodies using the chemiluminescent microparticle immunoassay method. This test does not inform on the genotypic characteristics of the virus or the actual presence of the virus in blood. This study therefore sought to determine the serologic and genotypic profiles of HBV circulating among the voluntary blood donors in Nairobi. METHODS: Blood samples were collected in plain and EDTA vacutainers and tested for the Hepatitis B surface antigen (HBsAg). HBV DNA was then extracted from plasma, its overlapping P/S gene amplified and sequenced. The resulting sequences were used to analyze for the circulating genotypes and mutations within the P and S genes. Bivariate statistical analysis was used to determine the association between demographic factors and HBV infection. RESULTS: A seroprevalence of 2.3% (n = 7) was reported. The age group 19-28 years was significantly associated with HBV infection. Nine samples were positive for HBV DNA; these included 2 HBsAg positive samples and 7 HBsAg negative samples. Genotype A, sub genotype A1 was found to be exclusively prevalent while a number of mutations were reported in the "a" determinant segment of the major hydrophilic region of the S gene associated with antibody escape. RT mutations including mutation rt181T in the P gene conferring resistance against Lamivudine and other ʟ-nucleoside drugs were detected. CONCLUSION: There is a high prevalence of occult HBV infections among these blood donors and therefore the testing platform currently in use requires revision.

Intra-host diversity of hepatitis B virus during mother-to-child transmission: the X gene may play a key role in virus survival in children after transmission.

Mother-to-child transmission of hepatitis B virus (HBV) is the main route of transmission in Asia, and characterization of HBV quasispecies is needed to further understand virus evolution and adaptation. To understand changes in HBV during mother-to-child transmission, we enrolled nine pairs of mothers and children in the study, including a set of twins. Three groups were infected with HBV genotype C, and six groups were infected with HBV genotype B. The full-length HBV genome was amplified by PCR from serum samples before antiviral treatment, the whole viral genomes from each pair were sequenced, and the complexity and diversity of the quasispecies were analyzed. The entropy of transmitted HBV in children was found to be lower than their mothers, suggesting that there was a bottleneck effect during HBV transmission from the mother to the child. Selective evolution was shown by calculating πN and πS in the whole genomes, and the highest values were obtained for the X gene, which plays a role in viral replication and immune escape. All genotype C patients and only one genotype B pair had a πN/πS greater than 1 ratio, indicating that positive selection had occurred. In addition, quasispecies were found to be different between the twin children despite having the same mother, indicating that virus evolution is host-specific.

Epidemiology of hepatitis B and C virus infection in Central West Argentina.

Little information is available regarding the prevalence of viral hepatitis in Central West Argentina. This study aims to give new information regarding HBV and HCV prevalence, genotypes, and risk factors in Central West Argentina and the suitability of dried blood spot (DBS) sampling for HBV and HCV screening. METHODS: A total of 622 individuals were included; the mean age was 36.6 ± 14.3 years and 55.4% were females. HBV and HCV markers were detected using serological and molecular analysis, and risk factors were evaluated using statistical analysis. RESULTS: Using serum samples, the HBsAg prevalence was 1.8%, the rate of HBV exposure (anti-HBc positivity) was 5.3%, and the rate of HBV immunity was 34.9%. HBV DNA was found in four out of 11 HBsAg+ samples, and the viruses in three of these samples were classified as genotypes A1, A2 and F2a. Multivariate analysis showed that anti-HBs positivity was associated with the level of schooling and history of HBV vaccination. The anti-HCV prevalence was 2.6%, and HCV RNA was found in 11 samples, seven of which contained viruses of genotypes 1a (n = 2), 1b (n = 3) and 2 (n = 2). The sensitivity of the DBS assay for HBsAg, anti-HBc, and anti-HCV was 100%, 66.6%, and 75%, respectively, and the specificity was above 98% for all markers when compared to serum. CONCLUSION: A low rate of HBV immunity was observed, demonstrating the importance of HBV vaccination. High HCV prevalence was found, and HCV 1b was closely related to other Argentinian isolates. Finally, the performance of DBS testing in this population needs more optimization to increase its sensitivity and specificity.

PreS1 Mutations Alter the Large HBsAg Antigenicity of a Hepatitis B Virus Strain Isolated in Bangladesh.

Mutations in the hepatitis B virus (HBV) genome can potentially lead to vaccination failure, diagnostic escape, and disease progression. However, there are no reports on viral gene expression and large hepatitis B surface antigen (HBsAg) antigenicity alterations due to mutations in HBV isolated from a Bangladeshi population. Here, we sequenced the full genome of the HBV isolated from a clinically infected patient in Bangladesh. The open reading frames (ORFs) (P, S, C, and X) of the isolated HBV strain were successfully amplified and cloned into a mammalian expression vector. The HBV isolate was identified as genotype C (sub-genotype C2), serotype adr, and evolutionarily related to strains isolated in Indonesia, Malaysia, and China. Clinically significant mutations, such as preS1 C2964A, reverse transcriptase domain I91L, and small HBsAg N3S, were identified. The viral P, S, C, and X genes were expressed in HEK-293T and HepG2 cells by transient transfection with a native subcellular distribution pattern analyzed by immunofluorescence assay. Western blotting of large HBsAg using preS1 antibody showed no staining, and preS1 ELISA showed a significant reduction in reactivity due to amino acid mutations. This mutated preS1 sequence has been identified in several Asian countries. To our knowledge, this is the first report investigating changes in large HBsAg antigenicity due to preS1 mutations.

Identification of hepatitis B virus genotype I in Thailand.

The rare hepatitis B virus genotype I (HBV-I) classification includes complex A/G/C/U recombinants identified amongst the individuals from China, India, Laos, and Vietnam. Herein we report the first HBV-I specimen from Thailand, with detectable HBsAg despite a 10-amino-acid truncation. This HBV-I genome has a similar recombinant pattern to reference strains, including a C region that branches basal to references, suggesting a premodern era recombination event gave rise to HBV-I. With an average sequence divergence from other genotypes ranging from 7.66% (standard deviation [SD], 0.42%; C) to 14.27% (SD, 0.31%; H), this new genome supports the HBV-I classification as a unique genotype.

Hepatitis B virus in Mar del Plata, Argentina: Genomic characterization and evolutionary analysis of subgenotype F1b.

The aim is to describe the molecular epidemiology and perform a genomic characterization of hepatitis B virus (HBV) circulating in Mar del Plata and to identify the origin and diversification patterns of the most prevalent genotype. The S gene and the region encompassing the X gene, basal core promoter (BCP), and precore (preC) was analyzed in 56 samples. They were genotyped as: 80% F1b, 9% A2, 7% D3, and 2% D1. A recombinant F4/D2 genome was detected. The double substitution G1764A/A1762T at the BCP (reduced HBeAg expression) was found in 20% F1b, 2% A2, 2% D1, and 2% D3 samples. A unique D3 presented the G1896A substitution at the preC (HBeAg negative phenotype). A 13% of the samples showed mutations at the HBsAg "a" immunodeterminant (escape from neutralizing antibodies). Mutations at the polymerase (antiviral resistance) were found in 52% of the samples. Coalescent analysis of subgenotype F1b, the most prevalent in the city, showed that viral diversification in Mar del Plata started by year 2000. F1b was the most prevalent genotype detected, being a characteristic of actual HBV infections in Mar del Plata. Local HBV exhibit clinically relevant mutations, but a minority of them was shown to be associated to potential vaccination escape or antiviral resistance. Nevertheless, further studies are needed to determine whether any of these mutants could pose a threat to prevention, diagnosis, or treatment.