Subgenotype A1 Research Articles

First Detection of Hepatitis B Virus Subgenotype A5, and Characterization of Occult Infection and Hepatocellular Carcinoma-Related Mutations in Latin American and African Immigrants in Brazil.

This study aims to characterize the molecular profile of the hepatitis B virus (HBV) among socially vulnerable immigrants residing in Brazil to investigate the introduction of uncommon HBV strains into the country. Serum samples from 102 immigrants with positive serology for the HBV core antibody (anti-HBc) were tested for the presence of HBV DNA by PCR assays. Among these, 24 were also positive for the HBV surface antigen (HBsAg). The full or partial genome was sequenced to determine genotype by phylogenetic analysis. Participants were from Haiti (79.4%), Guinea-Bissau (11.8%), Venezuela (7.8%), and Colombia (1%). Of the 21 HBV DNA-positive samples, subgenotypes A1 (52.4%), A5 (28.6%), E (9.5%), F2 (4.8%), and F3 (4.8%) were identified. Among the 78 HBsAg-negative participants, four were positive for HBV DNA, resulting in an occult HBV infection rate of 5.1%. Phylogenetic analysis suggested that most strains were likely introduced to Brazil by migration. Importantly, 80% of A5 sequences had the A1762T/G1764A double mutation, linked to an increased risk of hepatocellular carcinoma development. In conclusion, this study is the first report of HBV subgenotype A5 in Brazil, shedding new light on the diversity of HBV strains circulating in the country. Understanding the genetic diversity of HBV in immigrant communities can lead to better prevention and control strategies, benefiting both immigrants and wider society.

Impact of Hepatitis Delta Virus Infection on the Selection of Hepatitis B Surface Antigen Mutations.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences (n = 55) were included in our study ([HIV/HBV-positive (n = 50) and HIV/HBV/HDV-positive (n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations (L95W, W156Q, C221Y) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations (W199R, C221A, C221S, W223G) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

Comparative Proteomic Analysis of Huh7 Cells Transfected with Sub-Saharan African Hepatitis B Virus (Sub)genotypes Reveals Potential Oncogenic Factors.

In sub-Saharan Africa (SSA), the (sub)genotypes A1, D3, and E of the hepatitis B virus (HBV) prevail. Individuals infected with subgenotype A1 have a 4.5-fold increased risk of HCC compared to those infected with other (sub)genotypes. The effect of (sub)genotypes on protein expression and host signalling has not been studied. Mass spectrometry was used to analyse the proteome of Huh7 cells transfected with replication-competent clones. Proteomic analysis revealed significantly differentially expressed proteins between SSA (sub)genotypes. Different (sub)genotypes have the propensity to dysregulate specific host signalling pathways. Subgenotype A1 resulted in dysregulation within the Ras pathway. Ras-associated protein, RhoC, was significantly upregulated in cells transfected with subgenotype A1 compared to those transfected with other (sub)genotypes, on both a proteomic (>1.5-fold) and mRNA level (p < 0.05). Two of the main cellular signalling pathways involving RHOC, MAPK and PI3K/Akt/mTOR, regulate cell growth, motility, and survival. Downstream signalling products of these pathways have been shown to increase MMP2 and MMP9 expression. An extracellular MMP2 and MMP9 ELISA revealed a non-significant increase in MMP2 and MMP9 in the cells transfected with A1 compared to the other (sub)genotypes (p < 0.05). The upregulated Ras-associated proteins have been implicated as oncoproteins in various cancers and could contribute to the increased hepatocarcinogenic potential of A1.

Genetic variability of hepatitis B virus in acute and in different phases of chronic infection in Brazil

The selection pressure imposed by the host immune system impacts on hepatitis B virus (HBV) variability. This study evaluates HBV genetic diversity, nucleos(t)ide analogs resistance and HBsAg escape mutations in HBV patients under distinct selective pressures. One hundred and thirteen individuals in different phases of HBV infection were included: 13 HBeAg-positive chronic infection, 9 HBeAg-positive chronic hepatitis, 47 HBeAg-negative chronic infection (ENI), 29 HBeAg-negative chronic hepatitis (ENH) and 15 acute infected individuals. Samples were PCR amplified, sequenced and genetically analyzed for the overlapping POL/S genes. Most HBV carriers presented genotype A (84/113; 74.3%), subgenotype A1 (67/84; 79.7%), irrespective of group, followed by genotypes D (20/113; 17.7%), F (8/113; 7.1%) and E (1/113; 0.9%). Clinically relevant mutations in polymerase (tL180M/M204V) and in the Major Hydrophilic Region of HBsAg (sY100C, T118A/M, sM133T, sD144A and sG145R) were observed. Our findings, however, indicated that most polymorphic sites were located in the cytosolic loops (CYL1-2) and transmembrane domain 4 (TMD4) of HBsAg. Lower viral loads and higher HBV genetic diversity were observed in ENI and ENH groups (p < 0.001), suggesting that these groups are subjected to a higher selective pressure. Our results provide information on the molecular characteristics of HBV in a diverse clinical setting, and may guide future studies on the balance of HBV quasispecies at different stages of infection.

Hepatitis E Virus Infection in Voluntary Blood Donors in the Russian Federation.

Transfusion-transmitted hepatitis E virus (HEV) infection is an increasing concern in many countries. We investigated the detection rate of HEV viremia in blood donors in Russia. A total of 20,405 regular repetitive voluntary non-renumerated blood donors from two regions (Moscow and Belgorod) were screened for HEV RNA using the cobas® HEV test in mini-pools of six plasma samples. Samples from each reactive pool were tested individually. The average HEV RNA prevalence was 0.024% (95% CI: 0.01-0.05%), or 1 case per 4081 donations. No statistically significant differences in HEV RNA prevalence were observed between the two study regions. The PCR threshold cycle (Ct) values ranged from 25.0 to 40.5 in reactive pools, and from 20.9 to 41.4 in reactive plasma samples when tested individually. The HEV viremic donors had different antibody patterns. Two donor samples were reactive for both anti-HEV IgM and IgG antibodies, one sample was reactive for anti-HEV IgM and negative for anti-HEV IgG, and two samples were seronegative. At follow-up testing 6 months later, on average, four donors available for follow-up had become negative for HEV RNA and positive for anti-HEV IgG. The HEV ORF2 sequence belonging to HEV-3 sub-genotype 3a was obtained from one donor sample. The sequencing failed in the other four samples from viremic donors, presumably due to the low viral load. In conclusion, the HEV RNA detection rate in blood donors in Russia corresponds with data from other European countries, including those that implemented universal donor HEV screening. These data support the implementation of HEV RNA donor screening to reduce the risk of transfusion-transmitted HEV infection in Russia.

Surveillance of Erythrovirus B19 (B19V) in patients with acute febrile illness suspected of arboviruses in Mato Grosso do Sul state, Brazil.

Human Erythrovirus (parvovirus) B19 infection can produce symptoms similar to those produced by Dengue, Chikungunya, and Zika viruses, making clinical diagnosis difficult. The importance of erythrovirus B19 in human pathology has been increased and reported in numerous studies published globally. The B19V infection was investigated by real-time PCR in sera samples from patients with signs and symptoms related to classic arboviral symptoms. This study was conducted to provide information on the genetic diversity of Human Erythrovirus B19 (B19V) circulating in the state of Mato Grosso do Sul, Midwest region of Brazil, from 2017 to 2022. A total of 773 sera samples of patients with negative diagnostic results for Dengue, Chikungunya, and Zika, during the study period were analyzed. Erythrovirus DNA was found in 10.6% (82/773) of patients, among them 10 were pregnant women. Four samples were completely sequenced, and the other five partially, to genotype by phylogenetic reconstruction. All samples belong to worldwide dispersed genotype 1, subgenotype 1a. The findings of the study demonstrate the importance of including B19V in differential laboratory diagnosis for epidemiological purposes and appropriate patient management. The diagnosis for B19V should be performed, particularly among pregnant women, immunocompromised patients, and individuals with hemolytic diseases, given that the infection is more severe in these cases.

Contribution of Hepatitis D Virus in Liver Cancer Among Hepatitis B Virus Infected Populations of Central Africa: A Systematic Review and Meta-Analysis

Background and aims: Hepatocellular carcinoma (HCC) is a rampant disease in sub-Saharan Africa. Viral hepatitis is currently the most common factor in the occurrence of HCC in sub-Saharan Africa. Hepatitis B virus (HBV) causes liver disease which may or may not progress to HCC. In a dual infection with Hepatitis D virus (HDV), HCC tends to have a faster turn out. Our aim is to evaluate the influence of HDV on the risk of HCC and elaborate on factors favouring HDV infection. Methods: A review of available literature in English or French was conducted in major data bases with the search terms “liver disease”, “hepatocellular carcinoma”, “hepatitis delta and liver disease”, “hepatitis D virus infection”, “prevalence of HDV”, and “hepatitis D in Central Africa”, and each term was associated to a central African country. A meta-analysis of included publications was then performed. Results: The prevalence of HDV was 28% (95% CI). There was a higher risk of HCC in HBV-HDV dual infection (OR: 0.82 [95% CI: -0.01 to 1.65]) compared to that in HBV mono-infection. HDV genotype 1 was found to be the most prominent in central Africa, with HDV subgenotypes 1a and 1b dominating. Hepatitis delta was more prevalent in rural areas than in urban areas. Conclusion: Hepatitis D is highly prevalent in the HBV population of central Africa. The presence of HDV increases the risk of HCC. More research has to be carried out to understand how HDV speeds up liver disease and hence how to prevent it.

Hepatitis B virus infection in patients presenting for immunosuppressive cancer therapy with and without underlying HIV infection

IntroductionReactivation of hepatitis B virus (HBV) infection induced by immunosuppressive cancer therapy is associated with fulminant liver disease and death. While national guidelines recommend HBV screening and antiviral prophylaxis for patients with cancer prior to initiating immunosuppressive therapy, compliance with these measures is unclear. This study characterized the burden of HBV infection among patients diagnosed with gynecological or dermatological cancers, with or without underlying HIV infection, before initiating immunosuppressive therapy. MethodsBetween 2016 – 2018, we recruited study patients from the Dr George Mukhari Academic Hospital in Tshwane, South Africa. Demographic (age, sex) and clinical data (HIV test results, HIV antiviral regimen, type of cancer) were recorded using a standardized data collection form. All participants were tested for HBV surface antigen (HBsAg), and antibodies to the surface (anti-HBs) and core antigens (anti-HBc). For detection of HBV DNA, a nested polymerase chain reaction was used to amplify polymerase gene fragments which were Sanger-sequenced and analyzed using bioinformatics software. All statistical analyses were performed using R version 4.1.0 (2021-05-18) and R studio version 2022.07.2. ResultsStudy participants were predominantly female (96.3%, 103/107) with a median (IQR) age of 50 (17.5) years. Cervical cancer was the most frequent cancer diagnosis (72%). Over half (52.3%; 56/107) of the participants were HIV positive and all but four (92.9%) on highly active antiretroviral therapy at the time of enrollment. The prevalence of chronic hepatitis B in the study population was 11.2% [95% CI:6.2-19.1], increasing to 14.3% [95% CI:6.8-26.8] in the HIV positive sub-population. The overall prevalence of occult HBV infection was 20% [95% CI:12.8-29.7], 57.9% [95% CI:33.97-78.9] of whom tested negative for all serological markers. Phylogenetic inference showed that all polymerase gene sequences generated in this study were sub-genotype A2. Mutational analysis did not reveal any drug resistance-associated amino acid variations in this study. ConclusionThese findings suggest that chronic and occult HBV infections are more prevalent among cancer patients with or without underlying HIV infection compared to what has previously been reported for the general South African population. This underscores the need to scale-up universal HBV serological and molecular screening with timely institution of prophylaxis prior to initiating immunosuppressive cancer therapy.

The epidemiological and genetic characteristics of human parvovirus B19 in patients with febrile rash illnesses in China

To understand the epidemiological and genetic characteristics of B19V, a multiple-province surveillance of patients with febrile rash illnesses (FRIs) were conducted in China during 2009 ~ 2021. The clinical specimens of 3,820 FRI patients were collected and tested for B19V DNA. A total of 99 (2.59%) patients were positive for B19V, and 49 (49.49%) were children under 5 years old. B19V infections occurred throughout the year without obvious seasonal pattern. Ten NS1-VP1u sequences and seven genome sequences were obtained in this study, identified as subgenotype 1a. Combined with the globally representative genome sequences, no temporal and geographic clustering trends of B19V were observed, and there was no significant correlation between B19V sequences and clinical manifestations. The evolutionary rate of the B19V genome was 2.30 × 10–4 substitutions/site/year. The number of negative selection sites was higher than that of positive selection sites. It was the first to comprehensively describe the prevalence patterns and evolutionary characteristics of B19V in FRI patients in China. B19V played the role in FRI patients. Children under 5 years old were the main population of B19V infection. Subgenotype 1a was prevalent in FRI patients in China. B19V showed a high mutation rate, while negative selection acted on the genome.

Genetic Diversity of Hepatitis B and C Viruses Revealed by Continuous Surveillance from 2015 to 2021 in Gabon, Central Africa.

Viral hepatitis remains one of the largest public health concerns worldwide. Especially in Central Africa, information on hepatitis virus infections has been limited, although the prevalence in this region has been reported to be higher than the global average. To reveal the current status of hepatitis B and C virus (HBV and HCV) infections and the genetic diversity of the viruses, we conducted longitudinal surveillance in Gabon. We detected 22 HBV and 9 HCV infections in 2047 patients with febrile illness. Genetic analyses of HBV identified subgenotype A1 for the first time in Gabon and an insertion generating a frameshift to create an X-preC/C fusion protein. We also revealed that most of the detected HCVs belonged to the "Gabon-specific" HCV subtype 4e (HCV-4e), and the entire nucleotide sequence of the HCV-4e polyprotein was determined to establish the first reference sequence. The HCV-4e strains possessed resistance-associated substitutions similar to those of other HCV-4 strains, indicating that the use of direct-acting antiviral therapy may be complex. These results provide a better understanding of the current situation of hepatitis B and C virus infections in Central Africa and will help public health organizations develop effective countermeasures to eliminate chronic viral hepatitis in this region.

Identification of the Genotypes Circulating in the Ecuadorian Population Infected with the Hepatitis C Virus (HCV).

The hepatitis C virus (HCV) is responsible for 1.5 million new infections, and around 290 thousand deaths worldwide. 15 to 30% of the patients that go into a chronic phase of the disease will develop cirrhosis or hepatocellular carcinoma within 20 years and is the leading etiology for liver transplantation. HCV genetic characteristics display a remarkable genetic diversity, which divides HCV into 8 genotypes and 67 subgenotypes; the treatment and probability of chronic HCV depend on these genotypes and subgenotypes. In Ecuador, there is no available information regarding HCV genotypes and subgenotypes; therefore, this study aims to provide an overview of the main genotypes circulating in Ecuador. In a cross-sectional and descriptive study using the Ecuadorian Ministry of Health (MSP) registry of patients already diagnosed with Hepatitis C (HCV) between 2017 and 2019. From 51 patients identified by health ministry, blood samples from a total of 15 subjects (named HCV1 to HCV15) were collected using an appropriate venipuncture technique. Pandemic-related circumstances avoid reaching all patients identified by health ministry. After the amplification of 11 samples from patients living in the Ecuadorian territory, the genotypes of HCV obtained were distributed as follows: 6 samples corresponding to subgenotype 2b (54.5%), 2 samples corresponding to subgenotype 1a (18.2%), 2 samples corresponding to subgenotype 4d (18.2%) and 1 corresponding to sample 1b (9.1%). These results represent the first epidemiological approach to genotype distribution in Ecuador, and it contributes to better management of patients. We emphasize the importance of the development of better strategies from the Healthcare Ministry of Ecuador (MSP) for the identification, treatment and tracking of HCV patients.

Expanding the range of the respiratory infectome in Australian feedlot cattle with and without respiratory disease using metatranscriptomics

BackgroundBovine respiratory disease (BRD) is one of the most common diseases in intensively managed cattle, often resulting in high morbidity and mortality. Although several pathogens have been isolated and extensively studied, the complete infectome of the respiratory complex consists of a more extensive range unrecognised species. Here, we used total RNA sequencing (i.e., metatranscriptomics) of nasal and nasopharyngeal swabs collected from animals with and without BRD from two cattle feedlots in Australia.ResultsA high abundance of bovine nidovirus, influenza D, bovine rhinitis A and bovine coronavirus was found in the samples. Additionally, we obtained the complete or near-complete genome of bovine rhinitis B, enterovirus E1, bovine viral diarrhea virus (sub-genotypes 1a and 1c) and bovine respiratory syncytial virus, and partial sequences of other viruses. A new species of paramyxovirus was also identified. Overall, the most abundant RNA virus, was the bovine nidovirus. Characterisation of bacterial species from the transcriptome revealed a high abundance and diversity of Mollicutes in BRD cases and unaffected control animals. Of the non-Mollicutes species, Histophilus somni was detected, whereas there was a low abundance of Mannheimia haemolytica.ConclusionThis study highlights the use of untargeted sequencing approaches to study the unrecognised range of microorganisms present in healthy or diseased animals and the need to study previously uncultured viral species that may have an important role in cattle respiratory disease.9Ej9w1gXTXyGbc-DCpnuD_Video

Frequency of drug resistance and immune escape mutations in the hepatitis B virus genome detected in pregnant women in the Republic of Guinea

The aim of the work is to assess the prevalence of hepatitis B virus drug resistance mutations and immune escape mutations in pregnant women in the Republic of Guinea.&#x0D; Materials and methods. Blood plasma samples obtained from 480 pregnant women from different regions of the Republic of Guinea with laboratory-confirmed viral hepatitis B were studied. Nucleotide sequences for genotype identification and mutation detection were obtained using nested-PCR followed by Sanger sequencing, based on overlapping pairs of primers spanning the complete genome of the virus.&#x0D; Results and discussion. In the examined group, the viral genotype E was the most prevalent (92.92%) compared with subgenotypes A1 (1.67%), A3 (1.46%), D1 (0.63%), D2 (1.04%) and D3 (2.29%). Among the examined HBV-infected pregnant women, 188 (39.17%) had undetectable HBsAg. Drug resistance mutations were detected in 33 individuals, which amounted to 6.88%. The following mutations were found: S78T (27.27%), L80I (24.24%), S202I (15.15%), M204I/V (42.42%). The presence of polymorphic variants not described as drug resistant has also been shown in positions associated with the development of drug resistance to tenofovir, lamivudine, telbivudine and entecavir (L80F, S202I, M204R). When analyzing the MHR and the region of a determinant, mutations were detected in 318 (66.25%) of pregnant women. In 172 of them, which amounted to 54.09%, multiple mutations were found. The amino acid substitutions in 13 positions associated with HBsAg-negative hepatitis B and/or potentially affecting HBsAg antigenicity were identified.&#x0D; Conclusion. The high prevalence of immune escape and drug resistance mutations potentially associated with false-negative result of HBsAg screening, prophylaxis failure, and virological failure of therapy that has been identified among treatment naive pregnant women imposes a serious problem.

Immunoinformatic prediction of the pathogenicity of bovine viral diarrhea virus genotypes: implications for viral virulence determinants, designing novel diagnostic assays and vaccines development.

Bovine viral diarrhea virus (BVDV) significantly impacts the bovine industries, both dairy and beef sectors. BVDV can infect various domestic and wild animals, most notably cattle. The dynamic variations among BVDV serotypes due to the continuous genetic diversity, especially in BVDV1 (BVDV1), reduce the effectiveness of the currently available vaccines and reduce the specificity/sensitivity of the diagnostic assays. The development of novel, safe, and effective vaccines against BVDV requires deep knowledge of the antigenicity and virulence of the virus. Previous studies on the antigenicity and the virulence of BVDV serotypes have been mainly focused on one or a few BVDV proteins. While however, little is known about the orchestration of all BVDV in the context of viral virulence and immunogenicity. The main aim of the current study was to do a comparative computational evaluation of the immunogenicity, and virulence for all the encoded proteins of both BVDV1 and BVDV2 and their sub-genotypes. To achieve this goal, 11,737 protein sequences were retrieved from Virus Pathogen Resource. The analysis involved a total of 4,583 sequences after the removal of short sequences and those with unknown collection time. We used the MP3 tool to map the pathogenic proteins across different BVDV strains. The potential protective and the epitope motifs were predicted using the VaxiJen and EMBOSS antigen tools, respectively. The virulence prediction revealed that the NS4B proteins of both BVDV1 and BVDV2 likely have essential roles in BVDV virulence. Similarly, both the capsid (C) and the NS4-A proteins of BVDV1 and the Npro and P7 proteins of BVDV2 are likely important virulent factors. There was a clear trend of increasing predicted virulence with the progression of time in the case of BVDV1 proteins, but that was not the case for the BVDV2 proteins. Most of the proteins of the two BVDV serotypes possess antigens predicted immunogens except Npro, P7, and NS4B. However, the predicted antigenicity of the BVDV1 was significantly higher than that of BVDV2. Meanwhile, the predicted immunogenicity of the immunodominant-E2 protein has been decreasing over time. Based on our predicted antigenicity and pathogenicity studies of the two BVDV serotypes, the sub-genotypes (1a, 1f, 1k, 2a, and 2b) may represent ideal candidates for the development of future vaccines against BVDV infection in cattle. In summary, we identified some common differences between the two BVDV genotypes (BVDV1 and BVDV2) and their sub-genotypes regarding their protein antigenicity and pathogenicity. The data presented here will increase our understanding of the molecular pathogenesis of BVDV infection in cattle. It will also pave the way for developing some novel diagnostic assays and novel vaccines against BVDV in the near future.

Patterns of hepatitis b virus immune escape and pol/rt mutations across clinical cohorts of patients with genotypes a, e and occult hepatitis b infection in Nigeria: A multi-centre study

ABSTRACT Hepatitis B virus (HBV) immune escape and Pol/RT mutations account for HBV immunoprophylactic, therapeutic, and diagnostic failure globally. Little is known about circulating HBV immune escape and Pol/RT mutants in Nigeria. This study focused on narrowing the knowledge gap of the pattern and prevalence of the HBV mutants across clinical cohorts of infected patients in southwestern Nigeria. Ninety-five enrollees were purposively recruited across clinical cohorts of HBV-infected patients with HBsAg or anti-HBc positive serological outcome and occult HBV infection. Total DNA was extracted from patients’ sera. HBV S and Pol gene-specific nested PCR amplification was carried out. The amplicons were further sequenced for serotypic, genotypic, phylogenetic, and mutational analysis. HBV S and Pol genes were amplified in 60 (63.2%) and 19 (20%) of HBV isolates, respectively. All the sixty HBV S gene and 14 of 19 Pol gene sequences were exploitable. The ayw4 serotype was predominant (95%) while ayw1 serotype was identified in 5% of isolates. Genotype E predominates in 95% of sequences, while genotype A, sub-genotype A3 was observed in 5%. Prevalence of HBV IEMs in the “a” determinant region was 29%. Commonest HBV IEM was S113T followed by G145A and D144E. The Pol/RT mutations rtV214A and rtI163V among others were identified in this study. This study provided data on the occurrence of existing and new HBV IEMs and Pol gene mutations in Nigeria.

Before Direct-Acting Antivirals for Hepatitis C Virus: Evaluation of Core Protein R70Q and L/C91M Substitutions in Chronically Infected Brazilian Patients Unresponsive to IFN and/or RBV.

Although chronic hepatitis C has been effectively treated with direct-acting antivirals (DAAs), the use of conventional therapy with peg-interferon (Peg-IFN) or (predominantly) ribavirin (RBV), remains widespread. R70Q/H and L/C91M amino acid substitutions in the hepatitis C virus (HCV) core protein may modulate responses to IFN and/or RBV, and are associated with cirrhosis, hepatocellular carcinoma (HCC), insulin resistance, and liver steatosis. We evaluated the R70Q/H and L/C91M substitutions, clinical and epidemiological profiles, and risk factors of Brazilian patients chronically infected with HCV subgenotypes 1a and 1b (HCV-GT1a and HCV-GT1b) unresponsive to IFN and/or RBV therapy. Sequencing and pyrosequencing analyses and sociodemographic and clinical predictive variables were used to assess the relationship between R70Q/H and L/C91M substitutions. Leukocyte counts, ALT levels, and ALT/AST ratios were significantly reduced in treated individuals, but more of these patients had advanced fibrosis and cirrhosis. L91M was more prevalent (19.7%), occurring only in HCV-GT1b, followed by R70Q/P (11.5%) and R70P (1.4%). R70Q/P exhibited higher mean AST, ALT, and GGT values, whereas L91M showed higher mean GGT values. Pyrosequencing of the L91M position revealed mutant subpopulations in 43.75% of samples.

Phylogeography of hepatitis B virus: The role of Portugal in the early dissemination of HBV worldwide.

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

Molecular diversity of hepatitis B virus among pregnant women in Amhara National Regional State, Ethiopia.

Despite the availability of effective vaccines and treatments for hepatitis B virus (HBV), it continues to be a major public health problem in sub-Saharan Africa including Ethiopia. Routine screening for HBV in pregnant women is widely recommended, but there is lack of screening for HBV during pregnancy in Ethiopia. Therefore, this study aimed to assess viral load, and genetic diversity among pregnant women in the Amhara National Regional State, Ethiopia. Hepatitis B surface antigen (HBsAg) testing was performed on 1846 pregnant women, 85 of who tested positive were included in this study. HBV DNA was isolated from 85 positive sera, and the partial surface/polymerase gene was amplified and sequenced. HBV genotypes, sub-genotypes, serotypes and mutations in surface genes and polymerase were studied. Out of 85 pregnant women`s HBsAg positive sera, 59(69.4%) had detectable viral DNA. The median viral load was 3.4 log IU/ml ranging from 2.6 to7.6 and 46 samples were successfully sequenced and genotyped. Genotypes A and D were identified in 39 (84.8%) and 7 (15.2%); respectively. All genotype A isolates were further classified into sub-genotype A1 and serotype adw2 (84.8%) whereas genotype D isolates were further classified into three sub genotypes; 2 (4.3%) D2, 1(2.2%) D4, and 4 (8.7%) D10 with serotypes ayw2 (10.9%), and ayw3 (4.3%). There were 19 (41.3%) surface gene mutations in the major hydrophilic region (MHR). Six (13.1%) of them were discovered in MHR`s `a'-determinant region. Six polymerase gene mutations (13%) were identified. Genotype A was the predominant genotype in the Amhara National Regional State. The surface and polymerase gene mutations identified in this study may lead to immune therapy failure, diagnostics escape and drug resistance. Thus, the data generated in this study will contribute to the planning of HBV diagnosis, vaccination and treatment, and most importantly to the prevention of vertical transmission of HBV in Ethiopia. Therefore, further molecular studies on HBV are warranted and continuous surveillance is important for patient management and for the prevention and control of HBV infection in the country.

Genetic Diversity and Possible Origins of the Hepatitis B Virus in Siberian Natives.

A total of 381 hepatitis B virus (HBV) DNA sequences collected from nine groups of Siberian native populations were phylogenetically analyzed along with 179 HBV strains sampled in different urban populations of former western USSR republics and 50 strains from Central Asian republics and Mongolia. Different HBV subgenotypes predominated in various native Siberian populations. Subgenotype D1 was dominant in Altaian Kazakhs (100%), Tuvans (100%), and Teleuts (100%) of southern Siberia as well as in Dolgans and Nganasans (69%), who inhabit the polar Taimyr Peninsula. D2 was the most prevalent subgenotype in the combined group of Nenets, Komi, and Khants of the northern Yamalo-Nenets Autonomous Region (71%) and in Yakuts (36%) from northeastern Siberia. D3 was the main subgenotype in South Altaians (76%) and Buryats (40%) of southeastern Siberia, and in Chukchi (51%) of the Russian Far East. Subgenotype C2 was found in Taimyr (19%) and Chukchi (27%), while subgenotype A2 was common in Yakuts (33%). In contrast, D2 was dominant (56%) in urban populations of the former western USSR, and D1 (62%) in Central Asian republics and Mongolia. Statistical analysis demonstrated that the studied groups are epidemiologically isolated from each other and might have contracted HBV from different sources during the settlement of Siberia.