Natalia M. Araujo

High proportion of subgroup A′ (genotype A) among Brazilian isolates of Hepatitis B virus

Summary.Hepatitis B virus (HBV) genotype A has been divided recently into two subgroups, designated A-A′ (genotype A excluding A′) and A′. Isolates belonging to subgroup A′ have been identified in Africa. A new genotyping method, based on PCR amplification of the pre-S/S genome region and subsequent restriction fragment length polymorphism (RFLP) analysis, was developed, that established a correlation between RFLP subtypes and subgroups within genotype A. To investigate the occurrence of subgroup A′ in South America, 119 Brazilian HBV isolates were analyzed. Ninety-three (78%) of them belonged to genotype A, with three predominating RFLP subtypes: 44 (37%) isolates were classified as AI, 30 (25%) were AII, and 18 (15%) were AIII. Pre-S/S nucleotide sequences of 15 genotype A isolates were determined. Phylogenetic analysis performed with these 15 and an additional 41 sequences revealed that isolates AI and AII clustered in subgroup A′, whereas isolates AIII were classified into subgroup A-A′. The correlation RFLP subtypes-subgroups was confirmed by the presence of amino acid residues specific for subgroup A′ in the surface antigens and polymerase of isolates AI and AII. The high proportion (63%) of isolates from subgroup A′ suggested an African origin for a large number of Brazilian HBVs.

Hepatitis B virus infection from an evolutionary point of view: how viral, host, and environmental factors shape genotypes and subgenotypes

Hepatitis B virus (HBV) has an overwhelming distribution in the world and causes important human health problems. It has infected one-third of the global population and more than 350 million people are chronic carriers. Several aspects of HBV infection confer adaptive advantages that lead to a highly efficient dissemination of the virus through different routes of transmission. HBV genotypes and subgenotypes have been associated with differences in clinical and virological characteristics, indicating that they may play a role in the virus-host relationship. In particular, a clear association between genotype A and chronic outcomes in both children and adults depending on the subgenotype involved, and between genotype C and a higher risk of complications from HBV infection, has been demonstrated. Interestingly, subgenotype A2 and genotype C are respectively likely to predominate in high-risk groups for sexual transmission and in areas where perinatal transmission is the major mode of HBV dissemination. An evolutionary approach to HBV infection, based on the principles of natural selection, may offer explanations for how modes of transmission may favor some genotypes and subgenotypes over others and, ultimately, influence HBV virulence.

Occult hepatitis B virus infection in HIV-infected patients: Evaluation of biochemical, virological and molecular parameters.

Aim:  To determine the prevalence of occult hepatitis B virus (HBV) infection in a group of human immunodeficiency virus (HIV)‐infected Brazilian patients and to investigate its association with biochemical, virological and molecular features.

Hepatitis B virus intergenotypic recombinants worldwide: An overview

Novel variants generated by recombination events between different hepatitis B virus (HBV) genotypes have been increasingly documented worldwide, and the role of recombination in the evolutionary history of HBV is of significant research interest. In the present study, large-scale data retrieval and analysis on HBV intergenotypic recombinant genomes were performed. The geographical distribution of HBV recombinants as well as the molecular processes involved in recombination were examined. After review of published data, a total of 436 complete HBV sequences, previously identified as recombinants, were included in the recombination detection analysis. About 60% of HBV recombinants were B/C (n=179) and C/D (n=83) hybrids. A/B/C, A/C, A/C/G, A/D, A/E, A/G, B/C/U (U=unknown genotype), C/F, C/G, C/J, D/E, D/F, and F/G hybrids were additionally identified. HBV intergenotypic sequences were reported in almost all geographical regions with similar circulation patterns as their original genotypes, indicating the potential for spreading in a wide range of human populations and developing their own epidemiology. Recombination breakpoints were non-randomly distributed in the genome, and specific favored sites detected, such as within nt 1700-2000 and 2100-2300 regions, which displayed a statistically significant difference in comparison with the remaining genome. Elucidation of the effects of recombination events on the evolutionary history of HBV is critical to understand current and future evolution trends.

Hepatitis B viral markers in banked human milk before and after Holder pasteurization

BACKGROUND Blood screening for hepatitis B virus (HBV) is not universally performed for donor selection in human milk banks. OBJECTIVES To evaluate the frequency of detection of HBV surface antigen (HBsAg) and HBV-DNA in colostrum of HBV-infected nursing mothers before and after Holder pasteurization. STUDY DESIGN Forty-two concentrated breast milk samples were obtained within two postnatal weeks from 24 HBsAg-positive women (4 HBeAg-positive and 20 HBeAg-negative, anti-HBe-positive) were tested for the presence of HBsAg and HBV-DNA before and after Holder pasteurization (30min at 62.5 degrees C). RESULTS Before pasteurization, HBsAg and HBV-DNA were found in 14/24 (58%), and 20/24 (75%) first milk samples, respectively, obtained by 4 days after delivery. At least one marker was detected in 20/24 (83%) milk samples. Both markers were identified in milk of HBeAg-positive mothers, and most mothers with anti-HBe in blood had at least one HBV marker. Once detected, viral markers were frequently found in milk samples subsequently obtained from the same woman. Holder pasteurization did not affect the probability of detecting HBsAg (8/18, 44%), HBV-DNA (12/18, 67%), or at least one of them (15/18, 83%). CONCLUSIONS Although the biological implications of these findings remain to be determined, considering that HBV is highly contagious and most recipients of banked human milk are preterm infants, these findings should be taken into account when donors are enlisted for human milk banks without serological screening.

Identification of novel recombinants of hepatitis B virus genotypes F and G in human immunodeficiency virus-positive patients from Argentina and Brazil

Hepatitis B virus (HBV) genotype G (HBV/G) infection is almost always detected along with a co-infecting HBV strain that can supply HBeAg, typically HBV/A2. In this study we describe, in two human immunodeficiency virus (HIV)-positive patients from Argentina and Brazil, the first report of HBV/G infection in Argentina and co-circulation of HBV/G, HBV/F and G/F recombinants in the American continent. HBV isolates carrying the 36 bp insertion of HBV/G were the most prevalent in both patients, with >99 % of colonies hybridizing to a probe specific for this insertion. Phylogenetic analyses of full-length genomes and precore/core fragments revealed that F4 and F1b were the co-infecting subgenotypes in the Brazilian and Argentinian patients, respectively. Bootscanning analysis provided evidence of recombination in several clones from both patients, with recombination breakpoints located mainly at the precore/core region. These data should encourage further investigations on the clinical implications of HBV/G recombinants in HBV/HIV co-infected patients.

Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection

Small hepatitis B virus surface protein (S-HBsAg) variant Y100C has been associated with HBsAg-negative phenotype. To determine whether Y100C substitution yields impaired HBsAg or small amounts of HBsAg that may reduce HBsAg detection by commercial anti-HBsAg antibodies, two eukaryotic expression plasmids, one containing a wild-type S and the other an S gene from a Y100C variant, were constructed and their levels of HBsAg compared by ELISA after transfection of HuH7 cells. Unexpectedly, the extracellular HBsAg levels detected with Y100C plasmid were higher than those observed with the wild-type plasmid, but without statistical significance. We concluded that the Y100C substitution alone did not play a role in reducing HBsAg amounts or HBsAg affinity by commercial ELISA assay. Further studies on in vitro replication fitness with the complete genome of HBV isolates displaying or not Y100C substitution may elucidate whether this mutation affects HBV replication and consequently HBsAg production.

Phylogeography and evolutionary history of hepatitis B virus genotype F in Brazil

BackgroundHepatitis B virus (HBV) genotype F (HBV/F) is considered to be indigenous to the Americas, but its emergence and spread in the continent remain unknown. Previously, only two HBV/F complete genome sequences from Brazil were available, limiting the contribution of Brazilian isolates to the phylogenetic studies of HBV/F. The present study was carried out to assess the proportion and geographic distributions of HBV/F subgenotypes in Brazil, to determine the full-length genomic sequences of HBV/F isolates from different Brazilian geographic regions, and to investigate the detailed evolutionary history and phylogeography of HBV/F in Brazil.MethodsComplete HBV/F genomes isolated from 12 Brazilian patients, representing the HBV/F subgenotypes circulating in Brazil, were sequenced and analyzed together with sequences retrieved from GenBank, using the Bayesian coalescent and phylogeographic framework.ResultsPhylogenetic analysis using all Brazilian HBV/F S-gene sequences available in GenBank showed that HBV/F2a is found at higher frequencies countrywide and corresponds to all sequences isolated in the Brazilian Amazon Basin. In addition, the evolutionary analysis using complete genome sequences estimated an older median ancestral age for the Brazilian HBV/F2a compared to the Brazilian HBV/F1b and HBV/F4 subgenotypes, suggesting that HBV/F2a represents the original native HBV of Brazil. The phylogeographic patterns suggested a north-to-south flow of HBV/F2a from Venezuela to Brazil, whereas HBV/F1b and HBV/F4 strains appeared to have spread from Argentina to Brazil.ConclusionsThis study suggests a plausible route of introduction of HBV/F subgenotypes in Brazil and demonstrates the usefulness of recently developed computational tools for investigating the evolutionary history of HBV.

Lamivudine resistance and other mutations in the polymerase and surface antigen genes of hepatitis B virus associated with a fatal hepatic failure case

Background and Aim:  Resistance to lamivudine therapy of chronic hepatitis B virus (HBV) infection occurs by mutation in the YMDD motif of the reverse transcriptase (rt) domain (rtM204V/I) of the virus polymerase, and is usually accompanied by rtL180M mutation. Here we investigated virological factors associated with hepatic failure in a 58‐year‐old male, chronically HBV‐infected patient who died after 33 months of lamivudine therapy.

Expression of Hepatitis B virus surface antigen (HBsAg) from genotypes A, D and F and influence of amino acid variations related or not to genotypes on HBsAg detection.

The impact of hepatitis B virus (HBV) genotypes on the sensitivity of surface antigen (HBsAg) detection assays has been poorly investigated. Here, plasmids carrying consensus or variant coding sequences for HBV surface proteins from genotypes A, D and F, were constructed. HBsAg levels were evaluated in medium and extracts of transfected CHO cells by a commercial polyclonal-based assay. We show that HBsAg detection values of consensus forms from genotypes D and F were, respectively, 37% and 30% lower than those obtained by genotype A. However, the presence of two single variations, T143M in genotype A, and T125M in genotype D, produced a decrease of 44% and an increase of 34%, respectively, on HBsAg mean values in comparison with their consensus forms. In conclusion, HBsAg detection levels varied among HBV genotypes. However, unique amino acid substitutions not linked to genotypes, such as T125M and T143M described here, should have more implications in HBV immunological diagnostics than the set of variations characteristic of each HBV genotype.